Cytotoxic benzodiazepine derivatives

ABSTRACT

The invention relates to novel benzodiazepine derivatives with antiproliferative activity and more specifically to novel benzodiazepine compounds of formula (I)-(VII). The invention also provides conjugates of the benzodiazepine compounds linked to a cell-binding agent. The invention further provides compositions and methods useful for inhibiting abnormal cell growth or treating a proliferative disorder in a mammal using the compounds or conjugates of the invention.

REFERENCE TO RELATED APPLICATIONS

This application is a continuation application of U.S. Ser. No.13/397,195, filed on Feb. 15, 2012; which claims the benefit of thefiling dates, under 35 U.S.C. §119(e), of U.S. Provisional ApplicationNo. 61/443,062, filed on Feb. 15, 2011, and U.S. Provisional ApplicationNo. 61/483,499, filed on May 6, 2011, and U.S. Provisional ApplicationNo. 61/443,092, filed on Feb. 15, 2011, the entire contents of each ofwhich, including all drawings, formulae, specifications, and claims, areincorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates to novel cytotoxic compounds, andcytotoxic conjugates comprising these cytotoxic compounds andcell-binding agents. More specifically, this invention relates to novelbenzodiazepine compounds derivatives thereof, intermediates thereof,conjugates thereof, and pharmaceutically acceptable salts thereof, whichare useful as medicaments, in particular as anti-proliferative agents.

BACKGROUND OF THE INVENTION

Benzodiazepine derivatives are useful compounds for treating variousdisorders, and include medicaments such as, antiepileptlcs(imidazo[2,1-b][1,3,5]benzothiadiazepines, U.S. Pat. No. 4,444,688; U.S.Pat. No. 4,062,852), antibacterials(pyrimido[1,2-c][1,3,5]benzothiadiazepines, GB 1476684), diuretics andhypotensives (pyrrolo(1,2-b)[1,2,5]benzothiadiazepine 5,5 dioxide, U.S.Pat. No. 3,506,646), hypolipidemics (WO 03091232), anti-depressants(U.S. Pat. No. 3,453,266); osteoporosis (JP 2138272).

Recently, it has been shown in animal tumor models that benzodiazepinederivatives, such as pyrrolobenzodiazepines (PBDs), act as anti-tumoragents (N-2-imidazolyl alkyl substituted1,2,5-benzothiadiazepine-1,1-dioxide, U.S. Pat. No. 6,156,746),benzo-pyrido or dipyrido thiadiazepine (WO 2004/069843),pyrrolo[1,2-b][1,2,5]benzothiadiazepines andpyrrolo[1,2-b][1,2,5]benzodiazepine derivatives (WO2007/015280),tomaymycin derivatives (e.g., pyrrolo[1,4]benzodiazepines), such asthose described in WO 00/12508, WO2005/085260, WO2007/085930, and EP2019104. Benzodiazepines are also known to affect cell growth anddifferentiation (Kamal A., et al., Bioorg Med. Chem. 2008 Aug. 15;16(16):7804-10 (and references cited therein); Kumar R, Mini Rev Med.Chem. 2003 June; 3(4):323-39 (and references cited therein); Bednarski JJ, et al., 2004; Sutter A. P, et al., 2002; Blatt N B, et al., 2002),Kamal A. et al., Current Med. Chem., 2002; 2; 215-254, Wang J-J., J.Med. Chem., 2206; 49:1442-1449, Alley M. C. et al., Cancer Res. 2004;64:6700-6706, Pepper C. J., Cancer Res 2004; 74:6750-6755, Thurston D.E. and Bose D. S., Chem Rev 1994; 94:433-465; and Tozuka, Z., et al.,Journal of Antibiotics, (1983) 36; 1699-1708. General structure of PBDsis described in US Publication Number 20070072846. The PBDs differ inthe number, type and position of substituents, in both their aromatic Arings and pyrrolo C rings, and in the degree of saturation of the Cring. Their ability to form an adduct in the minor groove and crosslinkDNA enables them to interfere with DNA processing, hence their potentialfor use as antiproliferative agents.

The first pyrrolobenzodiazepine to enter the clinic, SJG-136 (NSC694501) is a potent cytotoxic agent that causes DNA inter-strandcrosslinks (S. G Gregson et al., 2001, J. Med. Chem., 44: 737-748; M. C.Alley et al., 2004, Cancer Res., 64: 6700-6706; J. A. Hartley et al.,2004, Cancer Res., 64: 6693-6699; C. Martin et al., 2005, Biochemistry.,44: 4135-4147; S. Arnould et al., 2006, Mol. Cancer. Ther., 5:1602-1509). Results from a Phase I clinical evaluation of SJG-136revealed that this drug was toxic at extremely low doses (maximumtolerated dose of 45 μg/m², and several adverse effects were noted,including vascular leak syndrome, peripheral edema, liver toxicity andfatigue. DNA damage was noted at all doses in circulating lymphocytes(D. Hochhauser et al., 2009, Clin. Cancer Res., 15: 2140-2147). Thus,there exists a need for improved benzodiazepine derivatives that areless toxic and still therapeutically active for treating a variety ofproliferative disease states, such as cancer.

SUMMARY OF THE INVENTION

Cytotoxic benzodiazepine dimers disclosed in the art possess two iminefunctionalities in their free form or reversibly protected form, such asa hydrate, alkoxylate or sulfonate. The presence of these two iminefunctionalities results in crosslinking of DNA (S. G. Gregson et al.,2001, J. Med. Chem., 44: 737-748). The present invention is partly basedon the unexpected finding that cell binding agent conjugates of newcytotoxic benzodiazepine derivatives, such as indolinobenzodiazapenedimers that are devoid of two imine functionalities (e.g., one iminefunctionality and one amine functionality), and thus incapable ofcrosslinking DNA, display a much higher therapeutic index (ratio ofmaximum tolerated dose to minimum effective dose) in vivo compared tobenzodiazepine derivatives that can crosslink DNA that are previouslydisclosed in the art.

Thus one object of the invention is to provide cytotoxic compoundcomprising a linking group with a reactive group bonded thereto capableof covalently linking the cytotoxic compound to a cell binding agent(CBA, see below), wherein the cytotoxic compound is represented by anyone of the following formulas:

or a pharmaceutically acceptable salt thereof, wherein:

-   -   the double line        between N and C represents a single bond or a double bond,        provided that when it is a double bond X is absent and Y is —H,        or a linear or branched alkyl having 1 to 4 carbon atoms, and        when it is a single bond, X is —H, the linking group with the        reactive group bonded thereto, or an amine protecting moiety;    -   Y is —H or a leaving group selected from —OR, —OCOR′, —OCOOR′,        —OCONR′R″, —NR′R″, —NR′COR″, —NR′NR′R″, an optionally        substituted 5 or 6-membered nitrogen-containing heterocycle        (e.g., piperidine, tetrahydropyrrole, pyrazole, morpholine,        etc.), a guanidinum represented by —NR′(C═NH)NR′R″, an amino        acid, or a peptide represented by —NRCOP′, wherein P′ is an        amino acid or a polypeptide containing between 2 to 20 amino        acid units, —SR, —SOR′, —SO₂M, —SO₃M, —OSO₃M, halogen, cyano and        an azido; or,    -   Y is a sulfite (HSO₃, HSO₂ or a salt of HSO₃ ⁻, SO₃ ²⁻ or HSO₂ ⁻        formed with a cation), metabisulfite (H₂S₂O₅ or a salt of S₂O₅        ²⁻ formed with a cation), mono-, di-, tri-, and        tetra-thiophosphate (PO₃SH₃, PO₂S₂H₂, POS₃H₂, PS₄H₂ or a salt of        PO₃S³⁻, PO₂S₂ ³⁻, POS₃ ³⁻ or PS₄ ³⁻ formed with a cation), thio        phosphate ester (R^(i)O)₂PS(OR^(i)), R^(i)S—, R^(i)SO, R^(i)SO₂,        R^(i)SO₃, thiosulfate (HS₂O₃ or a salt of S₂O₃ ²⁻ formed with a        cation), dithionite (HS₂O₄ or a salt of S₂O₄ ²⁻ formed with a        cation), phosphorodithioate (P(═S)(OR^(k′))(S)(OH) or a salt        thereof formed with a cation), hydroxamic acid (R^(k′)C(═O)NOH        or a salt formed with a cation), formaldehyde sulfoxylate        (HOCH₂SO₂ ⁻ or a salt of HOCH₂SO₂ ⁻ formed with a cation, such        as HOCH₂SO₂ ⁻Na⁺) or a mixture thereof, wherein R^(i) is a        linear or branched alkyl having 1 to 10 carbon atoms and is        substituted with at least one substituent selected from        —N(R^(j))₂, —CO₂H, —SO₃H, and —PO₃H; R^(i) can be further        optionally substituted with a substituent for an alkyl described        herein; R^(j) is a linear or branched alkyl having 1 to 6 carbon        atoms; R^(k′) is a linear, branched or cyclic alkyl, alkenyl or        alkynyl having 1 to 10 carbon atoms, aryl, heterocyclyl or        heteroaryl; preferably, Y is an adduct of a bisulfite, a        hydrosulfite, or a metabisulfite, or salts thereof (such as        sodium salt);    -   M is —H or a cation;    -   R, for each occurrence, is independently selected from the group        consisting of —H, an optionally substituted linear, branched or        cyclic alkyl, alkenyl or alkynyl having from 1 to 10 carbon        atoms, a polyethylene glycol unit —(CH₂CH₂O)_(n)—R^(c), an        optionally substituted aryl having 6 to 18 carbon atoms, an        optionally substituted 5- to 18-membered heteroaryl ring        containing one or more heteroatoms independently selected from        nitrogen, oxygen, and sulfur, or an optionally substituted 3- to        18-membered heterocyclic ring containing 1 to 6 heteroatoms        independently selected from O, S, N and P;    -   R′ and R″ are each independently selected from —H, —OH, —OR,        —NHR, —NR₂, —COR, an optionally substituted linear, branched or        cyclic alkyl, alkenyl or alkynyl having from 1 to 10 carbon        atoms, a polyethylene glycol unit —(CH₂CH₂O)_(n)—R^(c), and an        optionally substituted 3- to 18-membered heterocyclic ring        having 1 to 6 heteroatoms independently selected from O, S, N        and P;    -   R^(c) is —H or a substituted or unsubstituted linear or branched        alkyl having 1 to 4 carbon atoms, or the linking group with the        reactive group bonded thereto;    -   n is an integer from 1 to 24;    -   W is selected from C═O, C═S, CH₂, BH, SO and SO₂;    -   X′ is selected from —H, an amine-protecting group, the linking        group with the reactive group bonded thereto, an optionally        substituted linear, branched or cyclic alkyl, alkenyl or alkynyl        having from 1 to 10 carbon atoms, a polyethylene glycol unit        —(CH₂CH₂O)_(n)—R^(c), an optionally substituted aryl having 6 to        18 carbon atoms, an optionally substituted 5- to 18-membered        heteroaryl ring containing one or more heteroatoms independently        selected from nitrogen, oxygen, and sulfur, and an optionally        substituted 3- to 18-membered heterocyclic ring containing 1 to        6 heteroatoms independently selected from O, S, N and P;    -   Y′ is selected from —H, an oxo group, the linking group with the        reactive group bonded thereto, an optionally substituted linear,        branched or cyclic alkyl, alkenyl or alkynyl having from 1 to 10        carbon atoms, an optionally substituted 6- to 18-membered aryl,        an optionally substituted 5- to 18-membered heteroaryl ring        containing one or more heteroatoms independently selected from        nitrogen, oxygen, and sulfur, an optionally substituted 3 to        18-membered heterocyclic ring having 1 to 6 heteroatoms;    -   R₁, R₂, R₃, R₄, R₁′, R₂′, R₃′ and R₄′ are each independently        selected from the group consisting of —H, an optionally        substituted linear, branched or cyclic alkyl, alkenyl or alkynyl        having from 1 to 10 carbon atoms, a polyethylene glycol unit        —(OCH₂CH₂)_(n)—R^(c), halogen, guanidinium [—NH(C═NH)NH₂], —OR,        —NR′R″, —NO₂, —NCO, —NR′COR″, —SR, a sulfoxide represented by        —SOR′, a sulfone represented by —SO₂R′, a sulfonate —SO₃ ⁻M⁺, a        sulfate —OSO₃ ⁻M⁺, a sulfonamide represented by —SO₂NR′R″,        cyano, an azido, —COR′, —OCOR′, —OCONR′R″ and the linking group        with the reactive group bonded thereto;    -   R₆ is —H, —R, —OR, —SR, —NR′R″, —NO₂, halogen or the linking        group with the reactive group bonded thereto;    -   Z and Z′ are independently selected from —(CH₂)_(n′)—,        —(CH₂)_(n′)—CR₇R₈—(CH₂)_(na′)—, —(CH₂)_(n′)—NR₉—(CH₂)_(na′)—,        —(CH₂)_(n′)—O—(CH₂)_(na′)— and —(CH₂)_(n′)—S—(CH₂)_(na′)—;    -   n′ and na′ are the same or different, and are selected from 0,        1, 2 and 3;    -   R₇ and R₈ are the same or different, and are each independently        selected from —H, —OH, —SH, —COOH, —NHR′, a polyethylene glycol        unit —(OCH₂CH₂)_(n)—, an amino acid, a peptide unit bearing 2 to        6 amino acids, an optionally substituted linear, branched or        cyclic alkyl having from 1 to 10 carbon atoms;    -   R₉ is independently selected from —H, an optionally substituted        linear, branched or cyclic alkyl having from 1 to 10 carbon        atoms, a polyethylene glycol unit —(OCH₂CH₂)_(n)—;    -   A and A′ are the same or different, and are independently        selected from —O—, oxo (—C(═O)—), —CRR′O—, —CRR′—, —S—, —CRR′S—,        —NR₅ and —CRR′N(R₅)—;    -   R₅ for each occurrence is independently —H or an optionally        substituted linear or branched alkyl having 1 to 10 carbon        atoms;    -   D and D′ are the same or different, and are independently absent        or selected from the group consisting of an optionally        substituted linear, branched or cyclic alkyl, alkenyl or alkynyl        having 1 to 10 carbon atoms, an amino acid, a peptide bearing 2        to 6 amino acids, and a polyethylene glycol unit        (—OCH₂CH₂)_(n)—;    -   L is absent, the linking group with the reactive group bonded        thereto, a polyethylene glycol unit (—OCH₂CH₂)_(n)—, a linear,        branched or cyclic alkyl or alkenyl having 1 to 10 carbon atoms,        a phenyl group, a 3 to 18-membered heterocyclic ring or a 5- to        18-membered heteroaryl ring having 1 to 6 heteroatoms        independently selected from O, S, N and P, wherein the alkyl or        alkenyl is optionally substituted with the linking group with        the reactive group bonded thereto; phenyl or heterocyclic or        heteroaryl ring can be optionally substituted, wherein the        substituent can be the linking group with the reactive group        bonded thereto.

In certain embodiments, X is not the linking group with the reactivegroup bonded thereto. In certain embodiments, the double line

between N and C represents a single bond, Y is not —H.

In certain embodiments, the compound is not any one of the followingcompounds:

In certain embodiments, Y is a leaving group selected from —OR, —OCOR′,—OCOOR′, —OCONR′R″, —NR′R″, —NR′COR″, —NR′NR′R″, an optionallysubstituted 5- or 6-membered nitrogen-containing heterocycle (e.g.,piperidine, tetrahydropyrrole, pyrazole, morpholine, etc.), a guanidinumrepresented by —NR′(C═NH)NR′R″, an amino acid, or a peptide representedby —NRCOP′, wherein P′ is an amino acid or a polypeptide containingbetween 2 to 20 amino acid units, —SR, —SOR′, —SO₂M, —SO₃M, —OSO₃M,halogen, cyano and an azido.

A second object of the invention is to provide conjugates of cellbinding agents with the novel benzodiazepine compounds or derivativesthereof of the present invention. These conjugates are useful astherapeutic agents, which are delivered specifically to target cells andare cytotoxic.

Specifically, a conjugate of the invention may comprise: a cytotoxiccompound and a cell binding agent (CBA), wherein the cytotoxic compoundcomprises a linking group which covalently links the cytotoxic compoundto the CBA, and wherein the cytotoxic compound is represented by any oneof the following formulas:

or a pharmaceutically acceptable salt thereof, wherein:

-   -   the double line        between N and C represents a single bond or a double bond,        provided that when it is a double bond X is absent and Y is —H,        or a linear or branched alkyl having 1 to 4 carbon atoms, and        when it is a single bond, X is —H, the linking group, or an        amine protecting moiety;    -   Y is —H or a leaving group selected from —OR, —OCOR′, —OCOOR′,        —OCONR′R″, —NR′R″, —NR′COR″, —NR′NR′R″, an optionally        substituted 5 or 6-membered nitrogen-containing heterocycle        (e.g., piperidine, tetrahydropyrrole, pyrazole, morpholine), a        guanidinum represented by —NR′(C═NH)NR′R″, an amino acid, or a        peptide represented by —NRCOP′, wherein P′ is an amino acid or a        polypeptide containing between 2 to 20 amino acid units, —SR,        —SOR′, —SO₂M, —SO₃M, —OSO₃M, halogen, cyano and an azido; or,    -   Y is a sulfite (HSO₃, HSO₂ or a salt of HSO₃ ⁻, SO₃ ²⁻ or HSO₂ ⁻        formed with a cation), metabisulfite (H₂S₂O₅ or a salt of S₂O₅        ²⁻ formed with a cation), mono-, di-, tri-, and        tetra-thiophosphate (PO₃SH₃, PO₂S₂H₂, POS₃H₂, PS₄H₂ or a salt of        PO₃S³⁻, PO₂S₂ ³⁻, POS₃ ⁻ or PS₄ ³⁻ formed with a cation), thio        phosphate ester (R^(i)O)₂PS(OR^(i)), R^(i)S—, R^(i)SO, R^(i)SO₂,        R^(i)SO₃, thiosulfate (HS₂O₃ or a salt of S₂O₃ ²⁻ formed with a        cation), dithionite (HS₂O₄ or a salt of S₂O₄ ²⁻ formed with a        cation), phosphorodithioate (P(═S)(OR^(k′))(S)(OH) or a salt        thereof formed with a cation), hydroxamic acid (R^(k′)C(═O)NOH        or a salt formed with a cation), formaldehyde sulfoxylate        (HOCH₂SO₂ ⁻ or a salt of HOCH₂SO₂ ⁻ formed with a cation, such        as HOCH₂SO₂ ⁻Na⁺) or a mixture thereof, wherein R^(i) is a        linear or branched alkyl having 1 to 10 carbon atoms and is        substituted with at least one substituent selected from        —N(R^(j))₂, —CO₂H, —SO₃H, and —PO₃H; R^(i) can be further        optionally substituted with a substituent for an alkyl described        herein; R^(j) is a linear or branched alkyl having 1 to 6 carbon        atoms; R^(k′) is a linear, branched or cyclic alkyl, alkenyl or        alkynyl having 1 to 10 carbon atoms, aryl, heterocyclyl or        heteroaryl; preferably, Y is an adduct of a bisulfite, a        hydrosulfite, or a metabisulfite, or salts thereof (such as        sodium salt);    -   M is —H or a cation;    -   R, for each occurrence, is independently selected from the group        consisting of —H, an optionally substituted linear, branched or        cyclic alkyl, alkenyl or alkynyl having from 1 to 10 carbon        atoms, a polyethylene glycol unit —(CH₂CH₂O)_(n)—R^(c), an        optionally substituted aryl having 6 to 18 carbon atoms, an        optionally substituted 5- to 18-membered heteroaryl ring        containing one or more heteroatoms independently selected from        nitrogen, oxygen, and sulfur, or an optionally substituted 3- to        18-membered heterocyclic ring containing 1 to 6 heteroatoms        independently selected from O, S, N and P;    -   R′ and R″ are each independently selected from —H, —OH, —OR,        —NHR, —NR₂, —COR, an optionally substituted linear, branched or        cyclic alkyl, alkenyl or alkynyl having from 1 to 10 carbon        atoms, a polyethylene glycol unit —(CH₂CH₂O)_(n)—R^(c), and an        optionally substituted 3- to 18-membered heterocyclic ring        having 1 to 6 heteroatoms independently selected from O, S, N        and P;    -   R^(c) is —H or a substituted or unsubstituted linear or branched        alkyl having 1 to 4 carbon atoms, or the linking group;    -   n is an integer from 1 to 24;    -   W is selected from C═O, C═S, CH₂, BH, SO and SO₂;    -   X′ is selected from —H, an amine-protecting group, the linking        group, an optionally substituted linear, branched or cyclic        alkyl, alkenyl or alkynyl having from 1 to 10 carbon atoms, a        polyethylene glycol unit —(CH₂CH₂O)_(n)—R^(c), an optionally        substituted aryl having 6 to 18 carbon atoms, an optionally        substituted 5- to 18-membered heteroaryl ring containing one or        more heteroatoms independently selected from nitrogen, oxygen,        and sulfur, and an optionally substituted 3- to 18-membered        heterocyclic ring containing 1 to 6 heteroatoms independently        selected from O, S, N and P;    -   Y′ is selected from —H, an oxo group, the linking group, an        optionally substituted linear, branched or cyclic alkyl, alkenyl        or alkynyl having from 1 to 10 carbon atoms, an optionally        substituted 6- to 18-membered aryl, an optionally substituted 5-        to 18-membered heteroaryl ring containing one or more        heteroatoms independently selected from nitrogen, oxygen, and        sulfur, an optionally substituted 3 to 18-membered heterocyclic        ring having 1 to 6 heteroatoms;    -   R₁, R₂, R₃, R₄, R₁′, R₂′, R₃′ and R₄′ are each independently        selected from the group consisting of —H, an optionally        substituted linear, branched or cyclic alkyl, alkenyl or alkynyl        having from 1 to 10 carbon atoms, a polyethylene glycol unit        —(OCH₂CH₂)_(n)—R^(c), halogen, guanidinium [—NH(C═NH)NH₂], —OR,        —NR′R″, —NO₂, —NCO, —NR′COR″, —SR, a sulfoxide represented by        —SOR′, a sulfone represented by —SO₂R′, a sulfonate —SO₃ ⁻M⁺, a        sulfate —OSO₃ ⁻M⁺, a sulfonamide represented by —SO₂NR′R″,        cyano, an azido, —COR′, —OCOR′, —OCONR′R″ and the linking group;    -   R₆ is —H, —R, —OR, —SR, —NR′R″, —NO₂, halogen or the linking        group;    -   Z and Z′ are independently selected from —(CH₂)_(n′)—,        —(CH₂)_(n′)—CR₇R₈—(CH₂)_(na′)—, —(CH₂)_(n′)—NR₉—(CH₂)_(na′)—,        —(CH₂)_(n′)—O—(CH₂)_(na′)— and —(CH₂)_(n′)S—(CH₂)_(na′)—;    -   n′ and na′ are the same or different, and are selected from 0,        1, 2 and 3;    -   R₇ and R₈ are same or different, and are each independently        selected from —H, —OH, —SH, —COOH, —NHR′, a polyethylene glycol        unit —(OCH₂CH₂)_(n)—, an amino acid, a peptide unit bearing 2 to        6 amino acids, an optionally substituted linear, branched or        cyclic alkyl having from 1 to 10 carbon atoms;    -   R₉ is independently selected from —H, an optionally substituted        linear, branched or cyclic alkyl having from 1 to 10 carbon        atoms, a polyethylene glycol unit —(OCH₂CH₂)_(n)—;    -   A and A′ are the same or different, and are independently        selected from —O—, oxo (—C(═O)—), —CRR′O—, —CRR′—, —S—, —CRR′S—,        —NR₅ and —CRR′N(R₅)—,    -   R₅ for each occurrence is independently —H or an optionally        substituted linear or branched alkyl having 1 to 10 carbon        atoms;    -   D and D′ are the same or different, and are independently absent        or selected from the group consisting of an optionally        substituted linear, branched or cyclic alkyl, alkenyl or alkynyl        having 1 to 10 carbon atoms, an amino acid, a peptide bearing 2        to 6 amino acids, and a polyethylene glycol unit        (—OCH₂CH₂)_(n)—;    -   L is absent, the linking group, a polyethylene glycol unit        (—OCH₂CH₂)_(n)—, a linear, branched or cyclic alkyl or alkenyl        having 1 to 10 carbon atoms, a phenyl group, a 3- to 18-membered        heterocyclic ring or a 5- to 18-membered heteroaryl ring having        1 to 6 heteroatoms independently selected from O, S, N and P,        wherein the alkyl or alkenyl is optionally substituted with the        linking group; phenyl or heterocyclic or heteroaryl ring can be        optionally substituted, wherein the substituent can be the        linking group.

In certain embodiments, X is not the linking group. In certainembodiments, the double line

between N and C represents a single bond, Y is not —H.

In certain embodiments, Y is —H or a leaving group selected from —OR,—OCOR′, —OCOOR′, —OCONR′R″, —NR′R″, —NR′COR″, —NR′NR′R″, an optionallysubstituted 5- or 6-membered nitrogen-containing heterocycle (e.g.,piperidine, tetrahydropyrrole, pyrazole, morpholine, etc.), a guanidinumrepresented by —NR′(C═NH)NR′R″, an amino acid, or a peptide representedby —NRCOP′, wherein P′ is an amino acid or a polypeptide containingbetween 2 to 20 amino acid units, —SR, —SOR′, —SO₂M, —SO₃M, —OSO₃M,halogen, cyano and an azido. In certain embodiments, Y is not —H.

In certain embodiments, the compound of the conjugate is not any one ofthe following compounds (the wavy bond represents the bond through whichthe compound is linked to the CBA):

The present invention also includes a composition (e.g., apharmaceutical composition) comprising novel benzodiazepine compounds,derivatives thereof, or conjugates thereof, (and/or solvates, hydratesand/or salts thereof) and a carrier (a pharmaceutically acceptablecarrier). The present invention additionally includes a composition(e.g., a pharmaceutical composition) comprising novel benzodiazepinecompounds, derivatives thereof, or conjugates thereof (and/or solvates,hydrates and/or salts thereof), and a carrier (a pharmaceuticallyacceptable carrier), further comprising a second therapeutic agent. Thepresent compositions are useful for inhibiting abnormal cell growth ortreating a proliferative disorder in a mammal (e.g., human). The presentcompositions are useful for treating conditions such as cancer,rheumatoid arthritis, multiple sclerosis, graft versus host disease(GVHD), transplant rejection, lupus, myositis, infection, immunedeficiency such as AIDS, and inflammatory diseases in a mammal (e.g.,human).

The present invention includes a method of inhibiting abnormal cellgrowth or treating a proliferative disorder in a mammal (e.g., human)comprising administering to said mammal a therapeutically effectiveamount of novel benzodiazepine compounds, derivatives thereof, orconjugates thereof, (and/or solvates and salts thereof) or a compositionthereof, alone or in combination with a second therapeutic agent. Thepresent invention includes a method of synthesizing and using novelbenzodiazepine compounds, derivatives thereof, and conjugates thereoffor in vitro, in situ, and in vivo diagnosis or treatment of mammaliancells, organisms, or associated pathological conditions.

The compounds of this invention, derivatives thereof, or conjugatesthereof, and compositions comprising them, are useful for treating orlessening the severity of disorders, such as, characterized by abnormalgrowth of cells (e.g., cancer). Other applications for compounds andconjugates of this invention include, but are not limited to, treatingconditions such as cancer, rheumatoid arthritis, multiple sclerosis,graft versus host disease (GVHD), transplant rejection, lupus, myositis,infection, immune deficiency such as AIDS and inflammatory diseases in amammal (e.g., human).

As used herein, when referring to a group (e.g., R^(c), L, X′ etc.)“is/be” (or “is not”) the linking group or the linking group with thereactive group bounded thereto, it is meant that the group “comprises”(or “does not comprise”) the linking group or the linking group with thereactive group bounded thereto.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1-6 show the schemes for the synthesis of benzodiazepine compoundsand the corresponding linkable compounds suitable for conjugation of thepresent invention.

FIG. 7 shows the scheme for the synthesis of representative compoundswith PEG modified linkers of the present invention.

FIG. 8 shows the scheme for the synthesis of representative compoundswith a methylthio linker of the present invention.

FIGS. 9-10 show the schemes for the synthesis of representativecompounds containing a tertiary amine of the present invention.

FIG. 11 shows the scheme for the synthesis of representative compoundswith a peptide linker of the present invention.

FIGS. 12A, 12B, and 13-19 show the schemes for the synthesis ofrepresentative compounds suitable for one-step conjugation methods ofthe present invention.

FIG. 20 shows the scheme for a two-step mono-imine dimer synthesis.

FIG. 21 shows the scheme for a two-step di-reduced dimer synthesis.

FIG. 22 shows the scheme for the one-step synthesis of therepresentative antibody-drug conjugates.

FIG. 23 shows the scheme for the two-step synthesis of therepresentative antibody-drug conjugates.

FIG. 24 shows the in vitro cytotoxicity of the methyldithio dimer 1dagainst Namalwa, KB and HL60/QC cell lines.

FIG. 25 shows the in vitro cytotoxicity and specificity of thehuMy9-6-SPDB-1f conjugates against various cell lines. Note that sodiumbisulfite was added to the conjugation reaction for making theconjugate.

FIG. 26 shows the in vitro cytotoxicity and specificity of thehuFOLR1-SPDB-1f conjugates.

FIG. 27 shows conjugation of dimer does not reduce binding affinity ofantibody. Note that sodium bisulfite was added to the conjugationreaction for making the conjugate.

FIG. 28 shows the in vivo antitumor activity of huMy9-6 conjugate. Notethat sodium bisulfite was added to the conjugation reaction for makingthe conjugate.

FIG. 29 shows in vitro cytotoxicity of huMy9-6-SPDB-1f conjugate againstantigen positive cells. Note that sodium bisulfite was added to theconjugation reaction for making the conjugate.

FIG. 30 shows synthetic scheme for making thioether-containing linkerdisulfides 27e-h.

FIG. 31 shows synthetic scheme for making dimers 28c-f.

FIG. 32 shows synthetic scheme for making phenyl linked dimers 29b-c.

FIG. 33 shows the scheme for an alternative two-step synthesis formono-imine dimers.

FIG. 34 shows in vitro cytotoxicity for huMy9-6-SPDB-1f (A),huMy9-6-sulfoSPDB-1f (B) and huMy9-6-BMPS-1f (C) against HL60/QC (Ag⁺)cells with and without blocking of antigen binding sites. Note that inall three experiments (34A, 34B, and 34C), sodium bisulfite were addedto the conjugation reaction for making the conjugate.

FIG. 35 shows in vitro cytotoxicity for chB38.1-SPDB-1f (A), andchB38.1-sulfoSPDB-1f (B) against COLO205 (Ag⁺) cells. Note that in bothexperiments, sodium bisulfite was added to the conjugation reaction formaking the conjugate.

FIG. 36 shows in vivo efficacy of huMy9-6-SPDB-1f in HL60/QC bearingmice. Note that sodium bisulfite was added to the conjugation reaction.

FIG. 37 shows in vivo efficacy of huFOLR1-SPDB-1f in KB tumor bearingmice.

FIG. 38 shows synthetic scheme of compound 1.

FIG. 39 shows a synthetic scheme of compound 1d with5-ethyl-2-methylpyridine borane (PEMB).

FIG. 40 shows a synthetic scheme of compound 1d with sodiumtriacetoxyborohydride (STAB).

FIG. 41 shows a synthetic scheme of compound 31a-c.

FIG. 42 shows a synthetic scheme of compound 32c,d.

FIG. 43 shows a synthetic scheme of compounds 1i and 12a.

FIG. 44 shows antiproliferative activity by comparing (A)huMy9-6-SPDB-1f, (B) huMy9-6-sulfoSPDB-1f, and (C) huMy9-6-BMPS-1f,against OCI-AML3 (Ag⁺) cells with and without blocking of antigenbinding sites. Note that in all three experiments, sodium bisulfite wasadded to the conjugation reaction for making the conjugate.

FIG. 45 shows an alternate scheme for synthesizing4-(benzyloxy)-5-methoxy-2-nitrobenzoic acid used in the preparation ofIBD monomer.

FIG. 46 is an alternate synthesis scheme for(5-((2-(2-(2-methoxyethoxy)ethoxy)ethyl)(2-methyl-2-(methyldisulfanyl)propyl)amino)-1,3-phenylene)dimethanol(1b).

FIG. 47 is an alternate synthesis scheme for(5-((2-(2-(2-methoxyethoxy)ethoxy)ethyl)(2-methyl-2-(methyldisulfanyl)propyl)amino)-1,3-phenylene)dimethanol(1b).

FIG. 48 is an alternate synthetic scheme for a two-step mono-imine dimersynthesis.

FIG. 49 shows potency of various conjugates against various cell lines.The IC₅₀ values listed in the table are in the unit of nM.

FIG. 50 shows in vivo efficacy of huMy9-6-sulfo-SPDB-1f in MOLM-13 tumorbearing mice.

FIG. 51 shows in vivo efficacy of huMy9-6-sulfo-SPDB-1f in NB4 tumorbearing mice.

FIG. 52 shows in vivo efficacy of huMy9-6-BMPS-1f in HL60/QC tumorbearing mice.

FIG. 53 shows in vivo efficacy of huMy9-6-BMPS-1f in MOLM-13 tumorbearing mice. Note that sodium bisulfite was added to the conjugationreaction for making the conjugate.

FIG. 54 shows a representative synthesis scheme for a Sulfonatedfolate/cytotoxic compound conjugate.

FIG. 55 shows several representative sulfonated drug-antibody conjugateswith different linkers.

FIG. 56 shows in vivo efficacy of huMy9-6-Drug 2 in HL60/QC tumorbearing mice. Note that sodium bisulfite was added to the conjugationreaction for making the conjugate.

FIG. 57 shows in vivo efficacy of huMy9-6-Drug 2 in MOLM-13 tumorbearing mice. Note that sodium bisulfite was added to the conjugationreaction for making the conjugate.

FIG. 58 shows similar in vitro cytotoxicity of HuMy9-6-Drug 2(conjugates prepared without and with sodium bisulfite againstCD33-antigen expressing HL60 cells.

FIG. 59 shows similar in vitro cytotoxicity of anti-CD22 Ab-Drug 2conjugates prepared without and with sodium bisulfite againstCD22-antigen expressing BJAB cells.

FIG. 60 shows the preparation of huMy9-6-sulfo-SPDB-1d using the highlyreactive 4-nitroPy-sulfo-SPDB linker.

DETAILED DESCRIPTION OF THE INVENTION

Reference will now be made in detail to certain embodiments of theinvention, examples of which are illustrated in the accompanyingstructures and formulas. While the invention will be described inconjunction with the enumerated embodiments, it will be understood thatthey are not intended to limit the invention to those embodiments. Onthe contrary, the invention is intended to cover all alternatives,modifications, and equivalents which may be included within the scope ofthe present invention as defined by the claims. One skilled in the artwill recognize many methods and materials similar or equivalent to thosedescribed herein, which could be used in the practice of the presentinvention.

It should be understood that any of the embodiments described herein,including those described under different aspects of the invention(e.g., compounds, compound-linker molecules, conjugates, compositions,methods of making and using) and different parts of the specification(including embodiments described only in the Examples) can be combinedwith one or more other embodiments of the invention, unless explicitlydisclaimed or improper. Combination of embodiments are not limited tothose specific combinations claimed via the multiple dependent claims.

DEFINITIONS

“Linear or branched alkyl” as used herein refers to a saturated linearor branched-chain monovalent hydrocarbon radical of one to twenty carbonatoms. Examples of alkyl include, but are not limited to, methyl, ethyl,1-propyl, 2-propyl, 1-butyl, 2-methyl-1-propyl, —CH₂CH(CH₃)₂), 2-butyl,2-methyl-2-propyl, 1-pentyl, 2-pentyl 3-pentyl, 2-methyl-2-butyl,3-methyl-2-butyl, 3-methyl-1-butyl, 2-methyl-1-butyl, 1-hexyl), 2-hexyl,3-hexyl, 2-methyl-2-pentyl, 3-methyl-2-pentyl, 4-methyl-2-pentyl,3-methyl-3-pentyl, 2-methyl-3-pentyl, 2,3-dimethyl-2-butyl,3,3-dimethyl-2-butyl, 1-heptyl, 1-octyl, and the like. Preferably, thealkyl has one to ten carbon atoms. More preferably, the alkyl has one tofour carbon atoms.

“Linear or branched alkenyl” refers to linear or branched-chainmonovalent hydrocarbon radical of two to twenty carbon atoms with atleast one site of unsaturation, i.e., a carbon-carbon, double bond,wherein the alkenyl radical includes radicals having “cis” and “trans”orientations, or alternatively, “E” and “Z” orientations. Examplesinclude, but are not limited to, ethylenyl or vinyl (—CH═CH₂), allyl(—CH₂CH═CH₂), and the like. Preferably, the alkenyl has two to tencarbon atoms. More preferably, the alkyl has two to four carbon atoms.

“Linear or branched alkynyl” refers to a linear or branched monovalenthydrocarbon radical of two to twenty carbon atoms with at least one siteof unsaturation, i.e., a carbon-carbon, triple bond. Examples include,but are not limited to, ethynyl, propynyl, 1-butynyl, 2-butynyl,1-pentynyl, 2-pentynyl, 3-pentynyl, hexynyl, and the like. Preferably,the alkynyl has two to ten carbon atoms. More preferably, the alkynylhas two to four carbon atoms.

The term “carbocycle,” “carbocyclyl” and “carbocyclic ring” refer to amonovalent non-aromatic, saturated or partially unsaturated ring having3 to 12 carbon atoms as a monocyclic ring or 7 to 12 carbon atoms as abicyclic ring. Bicyclic carbocycles having 7 to 12 atoms can bearranged, for example, as a bicyclo [4,5], [5,5], [5,6], or [6,6]system, and bicyclic carbocycles having 9 or 10 ring atoms can bearranged as a bicyclo [5,6] or [6,6] system, or as bridged systems suchas bicyclo[2.2.1]heptane, bicyclo[2.2.2]octane and bicyclo[3.2.2]nonane.Examples of monocyclic carbocycles include, but are not limited to,cyclopropyl, cyclobutyl, cyclopentyl, 1-cyclopent-1-enyl,1-cyclopent-2-enyl, 1-cyclopent-3-enyl, cyclohexyl, 1-cyclohex-1-enyl,1-cyclohex-2-enyl, 1-cyclohex-3-enyl, cyclohexadienyl, cycloheptyl,cyclooctyl, cyclononyl, cyclodecyl, cycloundecyl, cyclododecyl, and thelike.

The terms “cyclic alkyl” and “cycloalkyl” can be used interchangeably.They refer to a monovalent saturated carbocyclic ring radical.Preferably, the cyclic alkyl is 3 to 7 membered monocyclic ring radical.More preferably, the cyclic alkyl is cyclohexyl.

The term “cyclic alkenyl” refers to a carbocyclic ring radical having atleast one double bond in the ring structure.

The term “cyclic alkynyl” refers to a carbocyclic ring radical having atleast one triple bond in the ring structure.

“Aryl” means a monovalent aromatic hydrocarbon radical of 6-18 carbonatoms derived by the removal of one hydrogen atom from a single carbonatom of a parent aromatic ring system. Some aryl groups are representedin the exemplary structures as “Ar.” Aryl includes bicyclic radicalscomprising an aromatic ring fused to a saturated, partially unsaturatedring, or aromatic carbocyclic or heterocyclic ring. Typical aryl groupsinclude, but are not limited to, radicals derived from benzene (phenyl),substituted benzenes, naphthalene, anthracene, indenyl, indanyl,1,2-dihydronapthalene, 1,2,3,4-tetrahydronapthyl, and the like.Preferably, aryl is phenyl group.

The terms “heterocycle,” “heterocyclyl,” and “heterocyclic ring” areused interchangeably herein and refer to a saturated or a partiallyunsaturated (i.e., having one or more double and/or triple bonds withinthe ring) carbocyclic radical of 3 to 18 ring atoms in which at leastone ring atom is a heteroatom selected from nitrogen, oxygen,phosphorus, and sulfur, the remaining ring atoms being C, where one ormore ring atoms is optionally substituted independently with one or moresubstituents described below. A heterocycle may be a monocycle having 3to 7 ring members (2 to 6 carbon atoms and 1 to 4 heteroatoms selectedfrom N, O, P, and S) or a bicycle having 7 to 10 ring members (4 to 9carbon atoms and 1 to 6 heteroatoms selected from N, O, P, and S), forexample: a bicyclo [4,5], [5,5], [5,6], or [6,6] system. Heterocyclesare described in Paquette, Leo A.; “Principles of Modern HeterocyclicChemistry” (W. A. Benjamin, New York, 1968), particularly Chapters 1, 3,4, 6, 7, and 9; “The Chemistry of Heterocyclic Compounds, A series ofMonographs” (John Wiley & Sons, New York, 1950 to present), inparticular Volumes 13, 14, 16, 19, and 28; and J. Am. Chem. Soc. (1960)82:5566. “Heterocyclyl” also includes radicals where heterocycleradicals are fused with a saturated, partially unsaturated ring, oraromatic carbocyclic or heterocyclic ring. Examples of heterocyclicrings include, but are not limited to, pyrrolidinyl, tetrahydrofuranyl,dihydrofuranyl, tetrahydrothienyl, tetrahydropyranyl, dihydropyranyl,tetrahydrothiopyranyl, piperidino, morpholino, thiomorpholino,thioxanyl, piperazinyl, homopiperazinyl, azetidinyl, oxetanyl,thietanyl, homopiperidinyl, oxepanyl, thiepanyl, oxazepinyl, diazepinyl,thiazepinyl, 2-pyrrolinyl, 3-pyrrolinyl, indolinyl, 2H-pyranyl,4H-pyranyl, dioxanyl, 1,3-dioxolanyl, pyrazolinyl, dithianyl,dithiolanyl, dihydropyranyl, dihydrothienyl, dihydrofuranyl,pyrazolidinylimidazolinyl, imidazolidinyl, 3-azabicyco[3.1.0]hexanyl,3-azabicyclo[4.1.0]heptanyl, and azabicyclo[2.2.2]hexanyl. Spiromoieties are also included within the scope of this definition. Examplesof a heterocyclic group wherein ring atoms are substituted with oxo (═O)moieties are pyrimidinonyl and 1,1-dioxo-thiomorpholinyl.

The term “heteroaryl” refers to a monovalent aromatic radical of 5- or6-membered rings, and includes fused ring systems (at least one of whichis aromatic) of 5-18 atoms, containing one or more heteroatomsindependently selected from nitrogen, oxygen, and sulfur. Examples ofheteroaryl groups are pyridinyl (including, for example,2-hydroxypyridinyl), imidazolyl, imidazopyridinyl, pyrimidinyl(including, for example, 4-hydroxypyrimidinyl), pyrazolyl, triazolyl,pyrazinyl, tetrazolyl, furyl, thienyl, isoxazolyl, thiazolyl, oxazolyl,isothiazolyl, pyrrolyl, quinolinyl, isoquinolinyl, indolyl,benzimidazolyl, benzofuranyl, cinnolinyl, indazolyl, indolizinyl,phthalazinyl, pyridazinyl, triazinyl, isoindolyl, pteridinyl, purinyl,oxadiazolyl, triazolyl, thiadiazolyl, furazanyl, benzofurazanyl,benzothiophenyl, benzothiazolyl, benzoxazolyl, quinazolinyl,quinoxalinyl, naphthyridinyl, and furopyridinyl.

The heterocycle or heteroaryl groups may be carbon (carbon-linked) ornitrogen (nitrogen-linked) attached where such is possible. By way ofexample and not limitation, carbon bonded heterocycles or heteroarylsare bonded at position 2, 3, 4, 5, or 6 of a pyridine, position 3, 4, 5,or 6 of a pyridazine, position 2, 4, 5, or 6 of a pyrimidine, position2, 3, 5, or 6 of a pyrazine, position 2, 3, 4, or 5 of a furan,tetrahydrofuran, thiofuran, thiophene, pyrrole or tetrahydropyrrole,position 2, 4, or 5 of an oxazole, imidazole or thiazole, position 3, 4,or 5 of an isoxazole, pyrazole, or isothiazole, position 2 or 3 of anaziridine, position 2, 3, or 4 of an azetidine, position 2, 3, 4, 5, 6,7, or 8 of a quinoline or position 1, 3, 4, 5, 6, 7, or 8 of anisoquinoline.

By way of example and not limitation, nitrogen bonded heterocycles orheteroaryls are bonded at position 1 of an aziridine, azetidine,pyrrole, pyrrolidine, 2-pyrroline, 3-pyrroline, imidazole,imidazolidine, 2-imidazoline, 3-imidazoline, pyrazole, pyrazoline,2-pyrazoline, 3-pyrazoline, piperidine, piperazine, indole, indoline,1H-indazole, position 2 of a isoindole, or isoindoline, position 4 of amorpholine, and position 9 of a carbazole, or O-carboline.

The heteroatoms present in heteroaryl or heterocycicyl include theoxidized forms such as NO, SO, and SO₂.

The term “halo” or “halogen” refers to F, Cl, Br or I.

The alkyl, alkenyl, alkynyl, cyclic alkyl, cyclic alkenyl, cyclicalkynyl, carbocyclyl, aryl, heterocyclyl and heteroaryl described abovecan be optionally substituted with one more (e.g., 2, 3, 4, 5, 6 ormore) substituents.

If a substituent is described as being “substituted,” a non-hydrogensubstituent is in the place of a hydrogen substituent on a carbon,oxygen, sulfur or nitrogen of the substituent. Thus, for example, asubstituted alkyl substituent is an alkyl substituent wherein at leastone non-hydrogen substituent is in the place of a hydrogen substituenton the alkyl substituent. To illustrate, monofluoroalkyl is alkylsubstituted with a fluoro substituent, and difluoroalkyl is alkylsubstituted with two fluoro substituents. It should be recognized thatif there is more than one substitution on a substituent, eachnon-hydrogen substituent may be identical or different (unless otherwisestated).

If a substituent is described as being “optionally substituted,” thesubstituent may be either (1) not substituted, or (2) substituted. If acarbon of a substituent is described as being optionally substitutedwith one or more of a list of substituents, one or more of the hydrogenson the carbon (to the extent there are any) may separately and/ortogether be replaced with an independently selected optionalsubstituent. If a nitrogen of a substituent is described as beingoptionally substituted with one or more of a list of substituents, oneor more of the hydrogens on the nitrogen (to the extent there are any)may each be replaced with an independently selected optionalsubstituent. One exemplary substituent may be depicted as —NR′R″,wherein R′ and R″ together with the nitrogen atom to which they areattached, may form a heterocyclic ring. The heterocyclic ring formedfrom R′ and R″ together with the nitrogen atom to which they areattached may be partially or fully saturated. In one embodiment, theheterocyclic ring consists of 3 to 7 atoms. In another embodiment, theheterocyclic ring is selected from the group consisting of pyrrolyl,imidazolyl, pyrazolyl, triazolyl, tetrazolyl, isoxazolyl, pyridyl andthiazolyl.

This specification uses the terms “substituent,” “radical,” and “group”interchangeably.

If a group of substituents are collectively described as beingoptionally substituted by one or more of a list of substituents, thegroup may include: (1) unsubstitutable substituents, (2) substitutablesubstituents that are not substituted by the optional substituents,and/or (3) substitutable substituents that are substituted by one ormore of the optional substituents.

If a substituent is described as being optionally substituted with up toa particular number of non-hydrogen substituents, that substituent maybe either (1) not substituted; or (2) substituted by up to thatparticular number of non-hydrogen substituents or by up to the maximumnumber of substitutable positions on the substituent, whichever is less.Thus, for example, if a substituent is described as a heteroaryloptionally substituted with up to 3 non-hydrogen substituents, then anyheteroaryl with less than 3 substitutable positions would be optionallysubstituted by up to only as many non-hydrogen substituents as theheteroaryl has substitutable positions. Such substituents, innon-limiting examples, can be selected from a linear, branched or cyclicalkyl, alkenyl or alkynyl having from 1 to 10 carbon atoms, aryl,heteroaryl, heterocycyclyl, halogen, guanidinium [—NH(C═NH)NH₂], —OR¹⁰⁰,NR¹⁰¹R¹⁰², —NO₂, —NR¹⁰¹COR¹⁰², —SR¹⁰⁰, a sulfoxide represented by—SOR¹⁰¹, a sulfone represented by —SO₂R¹⁰¹, a sulfonate —SO₃M, a sulfate—OSO₃M, a sulfonamide represented by —SO₂NR¹⁰¹R¹⁰², cyano, an azido,—COR¹⁰¹, —OCOR¹⁰¹, —OCONR¹⁰¹R¹⁰² and a polyethylene glycol unit(—OCH₂CH₂)_(n)R¹⁰¹ wherein M is H or a cation (such as Na⁺ or K⁺); R¹⁰²and R¹⁰³ are each independently selected from H, linear, branched orcyclic alkyl, alkenyl or alkynyl having from 1 to 10 carbon atoms, apolyethylene glycol unit (—OCH₂CH₂)_(n)—R¹⁰⁴, wherein n is an integerfrom 1 to 24, an aryl having from 6 to 10 carbon atoms, a heterocyclicring having from 3 to 10 carbon atoms and a heteroaryl having 5 to 10carbon atoms; and R¹⁰⁴ is H or a linear or branched alkyl having 1 to 4carbon atoms, wherein the alkyl, alkenyl, alkynyl, aryl, heteroaryl andheterocycicyl in the groups represented by R¹⁰⁰, R¹⁰¹, R¹⁰², R¹⁰³ andR¹⁰⁴ are optionally substituted with one or more (e.g., 2, 3, 4, 5, 6 ormore) substituents independently selected from halogen, —OH, —CN, —NO₂and unsubstituted linear or branched alkyl having 1 to 4 carbon atoms.Preferably, the substituents for the optionally substituted alkyl,alkenyl, alkynyl, cyclic alkyl, cyclic alkenyl, cyclic alkynyl,carbocyclyl, aryl, heterocyclyl and heteroaryl described above includehalogen, —CN, —NR¹⁰²R¹⁰³, —CF₃, —OR¹⁰¹, aryl, heteroaryl, heterocycycl,—SR¹⁰¹, —SOR¹⁰¹, —SO₂R¹⁰¹ and —SO₃M.

The term “compound” or “cytotoxic compound,” “cytotoxic dimer” and“cytotoxic dimer compound” are used interchangeably. They are intendedto include compounds for which a structure or formula or any derivativethereof has been disclosed in the present invention or a structure orformula or any derivative thereof that has been incorporated byreference. The term also includes, stereoisomers, geometric isomers,tautomers, solvates, metabolites, salts (e.g., pharmaceuticallyacceptable salts) and prodrugs, and prodrug salts of a compound of allthe formulae disclosed in the present invention. The term also includesany solvates, hydrates, and polymorphs of any of the foregoing. Thespecific recitation of “stereoisomers,” “geometric isomers,”“tautomers,” “solvates,” “metabolites,” “salt” “prodrug,” “prodrugsalt,” “conjugates,” “conjugates salt,” “solvate,” “hydrate,” or“polymorph” in certain aspects of the invention described in thisapplication shall not be interpreted as an intended omission of theseforms in other aspects of the invention where the term “compound” isused without recitation of these other forms.

The term “conjugate” as used herein refers to a compound describedherein or a derivative thereof that is linked to a cell binding agent.

The term “linkable to a cell binding agent” as used herein refers to thecompounds described herein or derivates thereof comprising at least onelinking group or a precursor thereof suitable to bond these compounds orderivatives thereof to a cell binding agent.

The term “precursor” of a given group refers to any group which may leadto that group by any deprotection, a chemical modification, or acoupling reaction.

The term “linked to a cell binding agent” refers to a conjugate moleculecomprising at least one of the compounds described herein (e.g.,compounds of formula (I)-(IV) and (VIII)-(XI) and drug-linker compoundsdescribe herein), or derivative thereof bound to a cell binding agentvia a suitable linking group or a precursor thereof.

The term “chiral” refers to molecules which have the property ofnon-superimposability of the mirror image partner, while the term“achiral” refers to molecules which are superimposable on their minorimage partner.

The term “stereoisomer” refers to compounds which have identicalchemical constitution and connectivity, but different orientations oftheir atoms in space that cannot be interconverted by rotation aboutsingle bonds.

“Diastereomer” refers to a stereoisomer with two or more centers ofchirality and whose molecules are not mirror images of one another.Diastereomers have different physical properties, e.g. melting points,boiling points, spectral properties, and reactivities. Mixtures ofdiastereomers may separate under high resolution analytical proceduressuch as crystallization, electrophoresis and chromatography.

“Enantiomers” refer to two stereoisomers of a compound which arenon-superimposable minor images of one another.

Stereochemical definitions and conventions used herein generally followS. P. Parker, Ed., McGraw-Hill Dictionary of Chemical Terms (1984)McGraw-Hill Book Company, New York; and Eliel, E. and Wilen, S.,“Stereochemistry of Organic Compounds,” John Wiley & Sons, Inc., NewYork, 1994. The compounds of the invention may contain asymmetric orchiral centers, and therefore exist in different stereoisomeric forms.It is intended that all stereoisomeric forms of the compounds of theinvention, including but not limited to, diastereomers, enantiomers andatropisomers, as well as mixtures thereof such as racemic mixtures, formpart of the present invention. Many organic compounds exist in optlcallyactive forms, i.e., they have the ability to rotate the plane ofplane-polarized light. In describing an optlcally active compound, theprefixes D and L, or R and S, are used to denote the absoluteconfiguration of the molecule about its chiral center(s). The prefixes dand I or (+) and (−) are employed to designate the sign of rotation ofplane-polarized light by the compound, with (−) or 1 meaning that thecompound is levorotatory. A compound prefixed with (+) or d isdextrorotatory. For a given chemical structure, these stereoisomers areidentical except that they are minor images of one another. A specificstereoisomer may also be referred to as an enantiomer, and a mixture ofsuch isomers is often called an enantiomeric mixture. A 50:50 mixture ofenantiomers is referred to as a racemic mixture or a racemate, which mayoccur where there has been no stereoselection or stereospecificity in achemical reaction or process. The terms “racemic mixture” and “racemate”refer to an equimolar mixture of two enantiomeric species, devoid ofoptlcal activity.

The term “tautomer” or “tautomeric form” refers to structural isomers ofdifferent energies which are interconvertible via a low energy barrier.For example, proton tautomers (also known as prototropic tautomers)include interconversions via migration of a proton, such as keto-enoland imine-enamine isomerizations. Valence tautomers includeinterconversions by reorganization of some of the bonding electrons.

The term “prodrug” as used in this application refers to a precursor orderivative form of a compound of the invention that is capable of beingenzymatically or hydrolytically activated or converted into the moreactive parent form. See, e.g., Wilman, “Prodrugs in Cancer Chemotherapy”Biochemical Society Transactions, 14, pp. 375-382, 615th Meeting Belfast(1986) and Stella et al., “Prodrugs: A Chemical Approach to TargetedDrug Delivery,” Directed Drug Delivery, Borchardt et al., (ed.), pp.247-267, Humana Press (1985). The prodrugs of this invention include,but are not limited to, ester-containing prodrugs, phosphate-containingprodrugs, thiophosphate-containing prodrugs, sulfate-containingprodrugs, peptide-containing prodrugs, D-amino acid-modified prodrugs,glycosylated prodrugs, β-lactam-containing prodrugs, optionallysubstituted phenoxyacetamide-containing prodrugs, optionally substitutedphenylacetamide-containing prodrugs, 5-fluorocytosine and other5-fluorouridine prodrugs which can be converted into the more activecytotoxic free drug. Examples of cytotoxic drugs that can be derivatizedinto a prodrug form for use in this invention include, but are notlimited to, compounds of the invention and chemotherapeutic agents suchas described above.

The term “prodrug” is also meant to include a derivative of a compoundthat can hydrolyze, oxidize, or otherwise react under biologicalconditions (in vitro or in vivo) to provide a compound of thisinvention. Prodrugs may only become active upon such reaction underbiological conditions, or they may have activity in their unreactedforms. Examples of prodrugs contemplated in this invention include, butare not limited to, analogs or derivatives of compounds of any one ofthe formulae disclosed herein that comprise biohydrolyzable moietiessuch as biohydrolyzable amides, biohydrolyzable esters, biohydrolyzablecarbamates, biohydrolyzable carbonates, biohydrolyzable ureides, andbiohydrolyzable phosphate analogues. Other examples of prodrugs includederivatives of compounds of any one of the formulae disclosed hereinthat comprise —NO, —NO₂, —ONO, or —ONO₂ moieties. Prodrugs can typicallybe prepared using well-known methods, such as those described byBurger's Medicinal Chemistry and Drug Discovery (1995) 172-178, 949-982(Manfred E. Wolff ed., 5th ed); see also Goodman and Gilman's, ThePharmacological basis of Therapeutics, 8th ed., McGraw-Hill, Int. Ed.1992, “Biotransformation of Drugs.”

One preferred form of prodrug of the invention includes compounds (withor without any linker groups) and conjugates of the invention comprisingan adduct formed between an imine bond of the compounds/conjugates andan imine reactive reagent. Another preferred form of prodrug of theinvention includes compounds such as those of formula (I)-(IV), whereinwhen the double line

between N and C represents a single bond, X is H or an amine protectinggroup, and the compound becomes a prodrug. A prodrug of the inventionmay contain one or both forms of prodrugs described herein (e.g.,containing an adduct formed between an imine bond of thecompounds/conjugates and an imine reactive reagent, and/or containing aY leaving group when X is —H).

The term “imine reactive reagent” refers to a reagent that is capable ofreacting with an imine group. Examples of imine reactive reagentincludes, but is not limited to, sulfites (H₂SO₃, H₂SO₂ or a salt ofHSO₃, SO₃ ² or HSO₂ formed with a cation), metabisulfite (H₂S₂O₅ or asalt of S₂O₅ ²⁻ formed with a cation), mono, di, tri, andtetra-thiophosphates (PO₃SH₃, PO₂S₂H₃, POS₃H₃, PS₄H₃ or a salt of PO₃S³,PO₂S₂ ³, POS₃ ³ or PS₄ ³⁻ formed with a cation), thio phosphate esters((R^(i)O)₂PS(OR^(i)), R^(i)SH, R^(i)SOH, R^(i)SO₂H, R^(i)SO₃H), variousamines (hydroxylamine (e.g., NH₂OH), hydrazine (e.g., NH₂NH₂),NH₂O—R^(i), R^(i), NH—R^(i), NH₂—R^(i)), NH₂—CO—NH₂, NH₂—C(═S)—NH₂′thiosulfate (H₂S₂O₃ or a salt of S₂O₃ ² formed with a cation),dithionite (H₂S₂O₄ or a salt of S₂O₄ ²⁻ formed with a cation),phosphorodithioate (P(═S)(OR^(k))(SH)(OH) or a salt thereof formed witha cation), hydroxamic acid (R^(k)C(═O)NHOH or a salt formed with acation), hydrazide (R^(k)CONHNH₂), formaldehyde sulfoxylate (HOCH₂SO₂Hor a salt of HOCH₂SO₂ ⁻ formed with a cation, such as HOCH₂SO₂ ⁻Na⁺),glycated nucleotide (such as GDP-mannose), fludarabine or a mixturethereof, wherein R^(i) and R^(i′) are each independently a linear orbranched alkyl having 1 to 10 carbon atoms and are substituted with atleast one substituent selected from —N(R^(j))₂, —CO₂H, —SO₃H, and —PO₃H;R^(i) and R^(i′) can be further optionally substituted with asubstituent for an alkyl described herein; R^(j) is a linear or branchedalkyl having 1 to 6 carbon atoms; and R^(k) is a linear, branched orcyclic alkyl, alkenyl or alkynyl having 1 to 10 carbon atoms, aryl,heterocyclyl or heteroaryl (preferably, R^(k) is a linear or branchedalkyl having 1 to 4 carbon atoms; more preferably, R^(k) is methyl,ethyl or propyl). Preferably, the cation is a monovalent cation, such asNa⁺ or K⁺. Preferably, the imine reactive reagent is selected fromsulfites, hydroxylamine, urea and hydrazine. More preferably, the iminereactive reagent is NaHSO₃ or KHSO₃.

As used herein and unless otherwise indicated, the terms“biohydrolyzable amide,” “biohydrolyzable ester,” “biohydrolyzablecarbamate,” “biohydrolyzable carbonate,” “biohydrolyzable ureide” and“biohydrolyzable phosphate analogue” mean an amide, ester, carbamate,carbonate, ureide, or phosphate analogue, respectively, that either: 1)does not destroy the biological activity of the compound and confersupon that compound advantageous properties in vivo, such as uptake,duration of action, or onset of action; or 2) is itself biologicallyinactive but is converted in vivo to a biologically active compound.Examples of biohydrolyzable amides include, but are not limited to,lower alkyl amides, α-amino acid amides, alkoxyacyl amides, andalkylaminoalkylcarbonyl amides. Examples of biohydrolyzable estersinclude, but are not limited to, lower alkyl esters, alkoxyacyloxyesters, alkyl acylamino alkyl esters, and choline esters. Examples ofbiohydrolyzable carbamates include, but are not limited to, loweralkylamines, substituted ethylenediamines, amino acids,hydroxyalkylamines, heterocyclic and heteroaromatic amines, andpolyether amines. Particularly favored prodrugs and prodrug salts arethose that increase the bioavailability of the compounds of thisinvention when such compounds are administered to a mammal.

The phrase “pharmaceutically acceptable salt” as used herein, refers topharmaceutically acceptable organic or inorganic salts of a compound ofthe invention. Exemplary salts include, but are not limited, to sulfate,citrate, acetate, oxalate, chloride, bromide, iodide, nitrate,bisulfate, phosphate, acid phosphate, isonicotinate, lactate,salicylate, acid citrate, tartrate, oleate, tannate, pantothenate,bitartrate, ascorbate, succinate, maleate, gentisinate, fumarate,gluconate, glucuronate, saccharate, formate, benzoate, glutamate,methanesulfonate “mesylate,” ethanesulfonate, benzenesulfonate,p-toluenesulfonate, pamoate (i.e.,1,1′-methylene-bis-(2-hydroxy-3-naphthoate)) salts, alkali metal (e.g.,sodium and potassium) salts, alkaline earth metal (e.g., magnesium)salts, and ammonium salts. A pharmaceutically acceptable salt mayinvolve the inclusion of another molecule such as an acetate ion, asuccinate ion or other counter ion. The counter ion may be any organicor inorganic moiety that stabilizes the charge on the parent compound.Furthermore, a pharmaceutically acceptable salt may have more than onecharged atom in its structure. Instances where multiple charged atomsare part of the pharmaceutically acceptable salt can have multiplecounter ions. Hence, a pharmaceutically acceptable salt can have one ormore charged atoms and/or one or more counter ion.

If the compound of the invention is a base, the desired pharmaceuticallyacceptable salt may be prepared by any suitable method available in theart, for example, treatment of the free base with an inorganic acid,such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid,methanesulfonic acid, phosphoric acid and the like, or with an organicacid, such as acetic acid, maleic acid, succinic acid, mandelic acid,fumaric acid, malonic acid, pyruvic acid, oxalic acid, glycolic acid,salicylic acid, a pyranosidyl acid, such as glucuronic acid orgalacturonic acid, an alpha hydroxy acid, such as citric acid ortartaric acid, an amino acid, such as aspartic acid or glutamic acid, anaromatic acid, such as benzoic acid or cinnamic acid, a sulfonic acid,such as p-toluenesulfonic acid or ethanesulfonic acid, or the like.

If the compound of the invention is an acid, the desiredpharmaceutically acceptable salt may be prepared by any suitable method,for example, treatment of the free acid with an inorganic or organicbase, such as an amine (primary, secondary or tertiary), an alkali metalhydroxide or alkaline earth metal hydroxide, or the like. Illustrativeexamples of suitable salts include, but are not limited to, organicsalts derived from amino acids, such as glycine and arginine, ammonia,primary, secondary, and tertiary amines, and cyclic amines, such aspiperidine, morpholine and piperazine, and inorganic salts derived fromsodium, calcium, potassium, magnesium, manganese, iron, copper, zinc,aluminum and lithium.

As used herein, the term “solvate” means a compound which furtherincludes a stoichiometric or non-stoichiometric amount of solvent suchas water, isopropanol, acetone, ethanol, methanol, DMSO, ethyl acetate,acetic acid, and ethanolamine dichloromethane, 2-propanol, or the like,bound by non-covalent intermolecular forces. Solvates or hydrates of thecompounds are readily prepared by addition of at least one molarequivalent of a hydroxylic solvent such as methanol, ethanol,1-propanol, 2-propanol or water to the compound to result in solvationor hydration of the imine moiety.

The terms “abnormal cell growth” and “proliferative disorder” are usedinterchangeably in this application. “Abnormal cell growth,” as usedherein, unless otherwise indicated, refers to cell growth that isindependent of normal regulatory mechanisms (e.g., loss of contactinhibition). This includes, for example, the abnormal growth of: (1)tumor cells (tumors) that proliferate by expressing a mutated tyrosinekinase or overexpression of a receptor tyrosine kinase; (2) benign andmalignant cells of other proliferative diseases in which aberranttyrosine kinase activation occurs; (3) any tumors that proliferate byreceptor tyrosine kinases; (4) any tumors that proliferate by aberrantserine/threonine kinase activation; and (5) benign and malignant cellsof other proliferative diseases in which aberrant serine/threoninekinase activation occurs.

The terms “cancer” and “cancerous” refer to or describe thephysiological condition in mammals that is typically characterized byunregulated cell growth. A “tumor” comprises one or more cancerouscells, and/or benign or pre-cancerous cells. Examples of cancer include,but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, andleukemia or lymphoid malignancies. More particular examples of suchcancers include squamous cell cancer (e.g., epithelial squamous cellcancer), lung cancer including small-cell lung cancer, non-small celllung cancer (“NSCLC”), adenocarcinoma of the lung and squamous carcinomaof the lung, cancer of the peritoneum, hepatocellular cancer, gastric orstomach cancer including gastrointestinal cancer, pancreatic cancer,glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladdercancer, hepatoma, breast cancer, colon cancer, rectal cancer, colorectalcancer, endometrial or uterine carcinoma, salivary gland carcinoma,kidney or renal cancer, prostate cancer, vulval cancer, thyroid cancer,hepatic carcinoma, anal carcinoma, penile carcinoma, acute leukemia,head/brain and neck cancer, cancers of lymphatic organs andhematological malignancy including Leukemia (Acute lymphoblasticleukemia (ALL), Acute myelogenous leukemia (AML), Chronic lymphocyticleukemia (CLL), Chronic myelogenous leukemia (CML), Acute monocyticleukemia (AMOL), Hairy cell leukemia (HCL), T-cell prolymphocyticleukemia (T-PLL), Large granular lymphocytic leukemia, Adult T-cellleukemia), Lymphoma (small lymphocytic lymphoma (SLL), Hodgkin'slymphomas (Nodular sclerosis, Mixed cellularity, Lymphocyte-rich,Lymphocyte depleted or not depleted, and Nodular lymphocyte-predominantHodgkin lymphoma), Non-Hodgkin's lymphomas (all subtypes), Chroniclymphocytic leukemia/Small lymphocytic lymphoma, B-cell prolymphocyticleukemia, Lymphoplasmacytic lymphoma (such as Waldenströmmacroglobulinemia), Splenic marginal zone lymphoma, Plasma cellneoplasms (Plasma cell myeloma, Plasmacytoma, Monoclonal immunoglobulindeposition diseases, Heavy chain diseases), Extranodal marginal zone Bcell lymphoma (MALT lymphoma), Nodal marginal zone B cell lymphoma(NMZL), Follicular lymphoma, Mantle cell lymphoma, Diffuse large B celllymphoma, Mediastinal (thymic) large B cell lymphoma, Intravascularlarge B cell lymphoma, Primary effusion lymphoma, Burkittlymphoma/leukemia, T cell prolymphocytic leukemia, T cell large granularlymphocytic leukemia, Aggressive NK cell leukemia, Adult T cellleukemia/lymphoma, Extranodal NK/T cell lymphoma (nasal type),Enteropathy-type T cell lymphoma, Hepatosplenic T cell lymphoma, BlasticNK cell lymphoma, Mycosis fungoides/Sezary syndrome, Primary cutaneousCD30-positive T cell lymphoproliferative disorders, Primary cutaneousanaplastic large cell lymphoma, Lymphomatoid papulosis,Angioimmunoblastic T cell lymphoma, Peripheral T cell lymphoma(unspecified), Anaplastic large cell lymphoma), multiple myeloma (plasmacell myeloma or Kahler's disease).

A “therapeutic agent” encompasses both a biological agent such as anantibody, a peptide, a protein, an enzyme or a chemotherapeutic agent.

A “chemotherapeutic agent” is a chemical compound useful in thetreatment of cancer. Examples of chemotherapeutic agents includeErlotinib (TARCEVA®, Genentech/OSI Pharm.), Bortezomib (VELCADE®,Millennium Pharm.), Fulvestrant (FASLODEX®, AstraZeneca), Sutent(SU11248, Pfizer), Letrozole (FEMARA®, Novartis), Imatinib mesylate(GLEEVEC®, Novartis), PTK787/ZK 222584 (Novartis), Oxaliplatin(Eloxatin®, Sanofi), 5-FU (5-fluorouracil), Leucovorin, Rapamycin(Sirolimus, RAPAMUNE®, Wyeth), Lapatinib (TYKERB®, GSK572016, GlaxoSmith Kline), Lonafarnib (SCH 66336), Sorafenib (BAY43-9006, BayerLabs), and Gefitinib (IRESSA®, AstraZeneca), AG1478, AG1571 (SU 5271;Sugen), alkylating agents such as thiotepa and CYTOXAN®cyclosphosphamide; alkyl sulfonates such as busulfan, improsulfan andpiposulfan; aziridines such as benzodopa, carboquone, meturedopa, anduredopa; ethylenimines and methylamelamines including altretamine,triethylenemelamine, triethylenephosphoramide,triethylenethiophosphoramide and trimethylomelamine; acetogenins(especially bullatacin and bullatacinone); a camptothecin (including thesynthetic analog topotecan); bryostatin; callystatin; CC-1065 (includingits adozelesin, carzelesin and bizelesin synthetic analogs);cryptophycins (particularly cryptophycin 1 and cryptophycin 8);dolastatin; duocarmycin (including the synthetic analogs, KW-2189 andCB1-TM1); eleutherobin; pancratistatin; a sarcodictyin; spongistatin;nitrogen mustards such as chlorambucil, chlornaphazine,chlorophosphamide, estramustine, ifosfamide, mechlorethamine,mechlorethamine oxide hydrochloride, melphalan, novembichin,phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosureassuch as carmustine, chlorozotocin, fotemustine, lomustine, nimustine,and ranimnustine; antibiotics such as the enediyne antibiotics (e.g.,calicheamicin, especially calicheamicin gammall and calicheamicinomegall (Angew Chem. Intl. Ed. Engl. (1994) 33:183-186); dynemicin,including dynemicin A; bisphosphonates, such as clodronate; anesperamicin; as well as neocarzinostatin chromophore and relatedchromoprotein enediyne antibiotic chromophores), aclacinomysins,actinomycin, authramycin, azaserine, bleomycins, cactinomycin,carabicin, caminomycin, carzinophilin, chromomycinis, dactinomycin,daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, ADRIAMYCIN®(doxorubicin), morpholino-doxorubicin, cyanomorpholino-doxorubicin,2-pyrrolino-doxorubicin and deoxydoxorubicin), epirubicin, esorubicin,idarubicin, marcellomycin, mitomycins such as mitomycin C, mycophenolicacid, nogalamycin, olivomycins, peplomycin, porfiromycin, puromycin,quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin,ubenimex, zinostatin, zorubicin; anti-metabolites such as methotrexateand 5-fluorouracil (5-FU); folic acid analogs such as denopterin,methotrexate, pteropterin, trimetrexate; purine analogs such asfludarabine, 6-mercaptopurine, thiamniprine, thioguanine; pyrimidineanalogs such as ancitabine, azacitidine, 6-azauridine, carmofur,cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine;androgens such as calusterone, dromostanolone propionate, epitiostanol,mepitiostane, testolactone; anti-adrenals such as aminoglutethimide,mitotane, trilostane; folic acid replenisher such as frolinic acid;aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil;amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine;diaziquone; elformithine; elliptinium acetate; an epothilone; etoglucid;gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids suchas maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol;nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone;podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK® polysaccharidecomplex (JHS Natural Products, Eugene, Oreg.); razoxane; rhizoxin;sizofuran; spirogermanium; tenuazonic acid; triaziquone;2,2′,2″-trichlorotriethylamine; trichothecenes (especially T-2 toxin,verracurin A, roridin A and anguidine); urethan; vindesine; dacarbazine;mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine;arabinoside (“Ara-C”); cyclophosphamide; thiotepa; taxoids, e.g., TAXOL®(paclitaxel; Bristol-Myers Squibb Oncology, Princeton, N.J.), ABRAXANE®(Cremophor-free), albumin-engineered nanoparticle formulations ofpaclitaxel (American Pharmaceutical Partners, Schaumberg, Ill.), andTAXOTERE® (doxetaxel; Rhone-Poulenc Rorer, Antony, France);chloranmbucil; GEMZAR® (gemcitabine); 6-thioguanine; mercaptopurine;methotrexate; platinum analogs such as cisplatin and carboplatin;vinblastine; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine;NAVELBINE® (vinorelbine); novantrone; teniposide; edatrexate;daunomycin; aminopterin; capecitabine (XELODA®); ibandronate; CPT-11;topoisomerase inhibitor RFS 2000; difluoromethylornithine (DMFO);retinoids such as retinoic acid; and pharmaceutically acceptable salts,acids and derivatives of any of the above.

Also included in the definition of “chemotherapeutic agent” are: (i)anti-hormonal agents that act to regulate or inhibit hormone action ontumors such as anti-estrogens and selective estrogen receptor modulators(SERMs), including, for example, tamoxifen (including NOLVADEX®;tamoxifen citrate), raloxifene, droloxifene, 4-hydroxytamoxifen,trioxifene, keoxifene, LY117018, onapristone, and FARESTON® (toremifinecitrate); (ii) aromatase inhibitors that inhibit the enzyme aromatase,which regulates estrogen production in the adrenal glands, such as, forexample, 4(5)-imidazoles, aminoglutethimide, MEGASE® (megestrolacetate), AROMASIN® (exemestane; Pfizer), formestanie, fadrozole,RIVISOR® (vorozole), FEMARA® (letrozole; Novartis), and ARIMIDEX®(anastrozole; AstraZeneca); (iii) anti-androgens such as flutamide,nilutamide, bicalutamide, leuprolide, and goserelin; as well astroxacitabine (a 1,3-dioxolane nucleoside cytosine analog); (iv) proteinkinase inhibitors; (v) lipid kinase inhibitors; (vi) antisenseoligonucleotides, particularly those which inhibit expression of genesin signaling pathways implicated in aberrant cell proliferation, suchas, for example, PKC-alpha, Ralf and H-Ras; (vii) ribozymes such as VEGFexpression inhibitors (e.g., ANGIOZYME®) and HER2 expression inhibitors;(viii) vaccines such as gene therapy vaccines, for example, ALLOVECTIN®,LEUVECTIN®, and VAXID®; PROLEUKIN® rIL-2; a topoisomerase 1 inhibitorsuch as LURTOTECAN®; ABARELIX® rmRH; (ix) anti-angiogenic agents such asbevacizumab (AVASTIN®, Genentech); and (x) pharmaceutically acceptablesalts, acids and derivatives of any of the above. Other anti-angiogenicagents include MMP-2 (matrix-metalloproteinase 2) inhibitors, MMP-9(matrix-metalloproteinase 9) inhibitors, COX-II (cyclooxygenase II)inhibitors, and VEGF receptor tyrosine kinase inhibitors. Examples ofsuch useful matrix metalloproteinase inhibitors that can be used incombination with the present compounds/compositions are described in WO96/33172, WO 96/27583, EP 818442, EP 1004578, WO 98/07697, WO 98/03516,WO 98/34918, WO 98/34915, WO 98/33768, WO 98/30566, EP 606,046, EP931,788, WO 90/05719, WO 99/52910, WO 99/52889, WO 99/29667, WO99/07675, EP 945864, U.S. Pat. No. 5,863,949, U.S. Pat. No. 5,861,510,and EP 780,386, all of which are incorporated herein in their entiretiesby reference. Examples of VEGF receptor tyrosine kinase inhibitorsinclude4-(4-bromo-2-fluoroanilino)-6-methoxy-7-(1-methylpiperidin-4-ylmethoxy)quinazoline(ZD6474; Example 2 within WO 01/32651),4-(4-fluoro-2-methylindol-5-yloxy)-6-methoxy-7-(3-pyrrolidin-1-ylpropoxy)-quinazoline(AZD2171; Example 240 within WO 00/47212), vatalanib (PTK787; WO98/35985) and SU11248 (sunitinib; WO 01/60814), and compounds such asthose disclosed in PCT Publication Nos. WO 97/22596, WO 97/30035, WO97/32856, and WO 98/13354).

Other examples of chemotherapeutic agents that can be used incombination with the present compounds include inhibitors of PI3K(phosphoinositide-3 kinase), such as those reported in Yaguchi et al(2006) Jour. of the Nat. Cancer Inst. 98(8):545-556; U.S. Pat. No.7,173,029; U.S. Pat. No. 7,037,915; U.S. Pat. No. 6,608,056; U.S. Pat.No. 6,608,053; U.S. Pat. No. 6,838,457; U.S. Pat. No. 6,770,641; U.S.Pat. No. 6,653,320; U.S. Pat. No. 6,403,588; WO 2006/046031; WO2006/046035; WO 2006/046040; WO 2007/042806; WO 2007/042810; WO2004/017950; US 2004/092561; WO 2004/007491; WO 2004/006916; WO2003/037886; US 2003/149074; WO 2003/035618; WO 2003/034997; US2003/158212; EP 1417976; US 2004/053946; JP 2001247477; JP 08175990; JP08176070; U.S. Pat. No. 6,703,414; and WO 97/15658, all of which areincorporated herein in their entireties by reference. Specific examplesof such PI3K inhibitors include SF-1126 (PI3K inhibitor, SemaforePharmaceuticals), BEZ-235 (PI3K inhibitor, Novartis), XL-147 (PI3Kinhibitor, Exelixis, Inc.).

Chemotherapeutic agents may also include any of the generic drugs orbiosimilars of the brand-name drugs referenced herein, or improvementsthereof, including improved formulations, prodrugs, delivery means(sustained release, bioadhesive coating, targeted delivery etc.), anddosage forms.

A “metabolite” is a product produced through metabolism in the body of aspecified compound, a derivative thereof, or a conjugate thereof, orsalt thereof. Metabolites of a compound, a derivative thereof, or aconjugate thereof, may be identified using routine techniques known inthe art and their activities determined using tests such as thosedescribed herein. Such products may result for example from theoxidation, hydroxylation, reduction, hydrolysis, amidation, deamidation,esterification, deesterification, enzymatic cleavage, and the like, ofthe administered compound. Accordingly, the invention includesmetabolites of compounds, a derivative thereof, or a conjugate thereof,of the invention, including compounds, a derivative thereof, or aconjugate thereof, produced by a process comprising contacting acompound, a derivative thereof, or a conjugate thereof, of thisinvention with a mammal for a period of time sufficient to yield ametabolic product thereof.

The phrase “pharmaceutically acceptable” indicates that the substance orcomposition must be compatible chemically and/or toxicologically, withthe other ingredients comprising a formulation, and/or the mammal beingtreated therewith.

The term “protecting group” or “protecting moiety” refers to asubstituent that is commonly employed to block or protect a particularfunctionality while reacting other functional groups on the compound, aderivative thereof, or a conjugate thereof. For example, an“amine-protecting group” or an “amino-protecting moiety” is asubstituent attached to an amino group that blocks or protects the aminofunctionality in the compound. Such groups are well known in the art(see for example P. Wuts and T. Greene, 2007, Protective Groups inOrganic Synthesis, Chapter 7, J. Wiley & Sons, NJ) and exemplified bycarbamates such as methyl and ethyl carbamate, FMOC, substituted ethylcarbamates, carbamates cleaved by 1,6-β-elimination (also termed “selfimmolative”), ureas, amides, peptides, alkyl and aryl derivatives.Suitable amino-protecting groups include acetyl, trifluoroacetyl,t-butoxycarbonyl (BOC), benzyloxycarbonyl (CBZ) and9-fluorenylmethylenoxycarbonyl (Fmoc). For a general description ofprotecting groups and their use, see P. G. M. Wuts & T. W. Greene,Protective Groups in Organic Synthesis, John Wiley & Sons, New York,2007.

The term “leaving group” refers to an group of charged or unchargedmoiety that departs during a substitution or displacement. Such leavinggroups are well known in the art and include, but not limited to,halogens, esters, alkoxy, hydroxyl, tosylates, triflates, mesylates,nitriles, azide, carbamate, disulfides, thioesters, thioethers anddiazonium compounds.

The term “bifunctional crosslinking agent,” “bifunctional linker” or“crosslinking agents” refers to modifying agents that possess tworeactive groups; one of which is capable of reacting with a cell bindingagent while the other one reacts with the cytotoxic compound to link thetwo moieties together. Such bifunctional crosslinkers are well known inthe art (see, for example, Isalm and Dent in Bioconjugation chapter 5,p218-363, Groves Dictionaries Inc. New York, 1999). For example,bifunctional crosslinking agents that enable linkage via a thioetherbond includeN-succinimidyl-4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (SMCC) tointroduce maleimido groups, or withN-succinimidyl-4-(iodoacetyl)-aminobenzoate (SIAB) to introduceiodoacetyl groups. Other bifunctional crosslinking agents that introducemaleimido groups or haloacetyl groups on to a cell binding agent arewell known in the art (see US Patent Applications 2008/0050310,20050169933, available from Pierce Biotechnology Inc. P.O. Box 117,Rockland, Ill. 61105, USA) and include, but not limited to,bis-maleimidopolyethyleneglycol (BMPEO), BM(PEO)₂, BM(PEO)₃,N-(β-maleimidopropyloxy)succinimide ester (BMPS), γ-maleimidobutyricacid N-succinimidyl ester (GMBS), E-maleimidocaproic acidN-hydroxysuccinimide ester (EMCS),5-maleimidovaleric acid NHS, HBVS,N-succinimidyl-4-(N-maleimidomethyl)-cyclohexane-1-carboxy-(6-amidocaproate),which is a “long chain” analog of SMCC (LC-SMCC),m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS),4-(4-N-maleimidophenyl)-butyric acid hydrazide or HCl salt (MPBH),N-succinimidyl 3-(bromoacetamido)propionate (SBAP), N-succinimidyliodoacetate (SIA), κ-maleimidoundecanoic acid N-succinimidyl ester(KMUA), N-succinimidyl 4-(p-maleimidophenyl)-butyrate (SMPB),succinimidyl-6-(β-maleimidopropionamido)hexanoate (SMPH),succinimidyl-(4-vinylsulfonyl)benzoate (SVSB), dithiobis-maleimidoethane(DTME), 1,4-bis-maleimidobutane (BMB), 1,4bismaleimidyl-2,3-dihydroxybutane (BMDB), bis-maleimidohexane (BMH),bis-maleimidoethane (BMOE), sulfosuccinimidyl4-(N-maleimido-methyl)cyclohexane-1-carboxylate (sulfo-SMCC),sulfosuccinimidyl(4-iodo-acetyl)aminobenzoate (sulfo-SIAB),m-maleimidobenzoyl-N-hydroxysulfosuccinimide ester (sulfo-MBS),N-(γ-maleimidobutryloxy)sulfosuccinimde ester (sulfo-GMBS),N-(ε-maleimidocaproyloxy)sulfosuccimido ester (sulfo-EMCS),N-(κ-maleimidoundecanoyloxy)sulfosuccinimide ester (sulfo-KMUS), andsulfosuccinimidyl 4-(p-maleimidophenyl)butyrate (sulfo-SMPB).

Heterobifunctional crosslinking agents are bifunctional crosslinkingagents having two different reactive groups. Heterobifunctionalcrosslinking agents containing both an amine-reactiveN-hydroxysuccinimide group (NHS group) and a carbonyl-reactive hydrazinegroup can also be used to link the cytotoxic compounds described hereinwith a cell-binding agent (e.g., antibody). Examples of suchcommercially available heterobifunctional crosslinking agents includesuccinimidyl 6-hydrazinonicotinamide acetone hydrazone (SANH),succinimidyl 4-hydrazidoterephthalate hydrochloride (SHTH) andsuccinimidyl hydrazinium nicotinate hydrochloride (SHNH). Conjugatesbearing an acid-labile linkage can also be prepared using ahydrazine-bearing benzodiazepine derivative of the present invention.Examples of bifunctional crosslinking agents that can be used includesuccinimidyl-p-formyl benzoate (SFB) andsuccinimidyl-p-formylphenoxyacetate (SFPA).

Bifunctional crosslinking agents that enable the linkage of cell bindingagent with cytotoxic compounds via disulfide bonds are known in the artand include N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP),N-succinimidyl-4-(2-pyridyldithio)pentanoate (SPP),N-succinimidyl-4-(2-pyridyldithio)butanoate (SPDB),N-succinimidyl-4-(2-pyridyldithio)₂-sulfo butanoate (sulfo-SPDB) tointroduce dithiopyridyl groups. Other bifunctional crosslinking agentsthat can be used to introduce disulfide groups are known in the art andare disclosed in U.S. Pat. Nos. 6,913,748, 6,716,821 and US PatentPublications 20090274713 and 20100129314, all of which are incorporatedherein by reference. Alternatively, crosslinking agents such as2-iminothiolane, homocysteine thiolactone or S-acetylsuccinic anhydridethat introduce thiol groups can also be used.

A “linker,” “linker moiety,” or “linking group” as defined herein refersto a moiety that connects two groups, such as a cell binding agent and acytotoxic compound, together. Typically, the linker is substantiallyinert under conditions for which the two groups it is connecting arelinked. A bifunctional crosslinking agent may comprise two reactivegroups, one at each ends of a linker moiety, such that one reactivegroup can be first reacted with the cytotoxic compound to provide acompound bearing the linker moiety and a second reactive group, whichcan then react with a cell binding agent. Alternatively, one end of thebifunctional crosslinking agent can be first reacted with the cellbinding agent to provide a cell binding agent bearing a linker moietyand a second reactive group, which can then react with a cytotoxiccompound. The linking moiety may contain a chemical bond that allows forthe release of the cytotoxic moiety at a particular site. Suitablechemical bonds are well known in the art and include disulfide bonds,thioether bonds, acid labile bonds, photolabile bonds, peptidase labilebonds and esterase labile bonds (see for example U.S. Pat. Nos.5,208,020; 5,475,092; 6,441,163; 6,716,821; 6,913,748; 7,276,497;7,276,499; 7,368,565; 7,388,026 and 7,414,073). Preferred are disulfidebonds, thioether and peptidase labile bonds. Other linkers that can beused in the present invention include non-cleavable linkers, such asthose described in are described in detail in U.S. publication number20050169933, or charged linkers or hydrophilic linkers and are describedin US 2009/0274713, US 2010/01293140 and WO 2009/134976, each of whichis expressly incorporated herein by reference, each of which isexpressly incorporated herein by reference.

In one embodiment, the linking group with a reactive group attached atone end, such as a reactive ester, is selected from the following:—O(CR₂₀R₂₁)_(m)(CR₂₂R₂₃)_(n)(OCH₂CH₂)_(p)(CR₄₀R₄₁)_(p″)Y″(CR₂₄R₂₅)_(q)(CO)_(t)X″,—O(CR₂₀R₂₁)_(m)(CR₂₆═CR₂₇)_(m′)(CR₂₂R₂₃)_(n)(OCH₂CH₂)_(p)(CR₄₀R₄₁)_(p″)Y″(CR₂₄R₂₅)_(q)(CO)_(t)X″,—O(CR₂₀R₂₁)_(m)(alkynyl)_(m′)(CR₂₂R₂₃)_(n)(OCH₂CH₂)_(p)(CR₄₀R₄₁)_(p)—Y″(CR₂₄R₂₅)_(q)(CO)_(t)X″,—O(CR₂₀R₂₁)_(m)(piperazino)_(t′)(CR₂₂R₂₃)_(n)(OCH₂CH₂)_(p)(CR₄₀R₄₁)_(p″)Y″(CR₂₄R₂₅)_(q)(CO)_(t)X″,—O(CR₂₀R₂₁)_(m)(pyrrolo)_(c)(CR₂₂R₂₃)_(n)(OCH₂CH₂)_(p)(CR₄₀R₄₁)_(p″)Y″(CR₂₄R₂₅)_(q)(CO)_(t)X″,—O(CR₂₀R₂₁)_(m)A″_(m″)(CR₂₂R₂₃)_(n)(OCH₂CH₂)_(p)(CR₄₀R₄₁)_(p″)Y″(CR₂₄R₂₅)_(q)(CO)_(t)X″,—S(CR₂₀R₂₁)_(m)(CR₂₂R₂₃)_(n)(OCH₂CH₂)_(p)(CR₄₀R₄₁)_(p″)Y″(CR₂₄R₂₅)_(q)(CO)_(t)X″,—S(CR₂₀R₂₁)_(m)(CR₂₆═CR₂₇)_(m′)(CR₂₂R₂₃)_(n)(OCH₂CH₂)_(p)(CR₄₀R₄₁)_(p″)Y″(CR₂₄R₂₅)_(q)(CO)_(t)X″,—S(CR₂₀R₂₁)_(m)(alkynyl)_(n′)(CR₂₂R₂₃)_(n)(OCH₂CH₂)_(p)(CR₄₀R₄₁)_(p″)Y″(CR₂₄R₂₅)_(q)(CO)_(t)X″,—S(CR₂₀R₂₁)_(m)(piperazino)_(t′)(CR₂₂R₂₃)_(n)(OCH₂CH₂)_(p)(CR₄₀R₄₁)_(p″)Y″(CR₂₄R₂₅)_(q)(CO)_(t)X″,—S(CR₂₀R₂₁)_(m)(pyrrolo)_(t′)(CR₂₂R₂₃)_(n)(OCH₂CH₂)_(p)(CR₄₀R₄₁)_(p″)Y″(CR₂₄R₂₅)_(q)(CO)_(t)X″,—S(CR₂₀R₂₁)_(m)A″_(m″)(CR₂₂R₂₃)_(n)(OCH₂CH₂)_(p)(CR₄₀R₄₁)_(p″)Y″(CR₂₄R₂₅)_(q)(CO)_(t)X″,—NR₃₃(C═O)_(p″)(CR₂₀R₂₁)_(m)(CR₂₂R₂₃)_(n)(OCH₂CH₂)_(p)(CR₄₀R₄₁)_(p″)Y″(CR₂₄R₂₅)_(q)(CO)_(t)X″,—NR₃₃(C═O)_(p″)(CR₂₀R₂₁)_(m)(CR₂₆═CR₂₇)_(a)(CR₂₂R₂₃)_(n)(OCH₂CH₂)_(p)(CR₄₀R₄₁)_(p″)Y″(CR₂₄R₂₅)_(q)(CO)_(t)X″,—NR₃₃(C═O)_(p″)(CR₂₀R₂₁)_(m)(alkynyl)_(n′)(CR₂₂R₂₃)_(n)(OCH₂CH₂)_(p)(CR₄₀R₄₁)_(p″)Y″(CR₂₄R₂₅)_(q)(CO)_(t)X″,—NR₃₃(CR₂₀R₂₁)_(p″)(CR₂₀R₂₁)_(m)(piperazino)_(t′)(CR₂₂R₂₃)_(n)(OCH₂CH₂)_(p)(CR₄₀R₄₁)_(p″)Y″(CR₂₄R₂₅)_(q)(CO)_(t)X″,—NR₃₃(C═O)_(p″)(CR₂₀R₂₁)_(m)(pyrrolo)_(t′)(CR₂₂R₂₃)_(n)(OCH₂CH₂)_(p)(CR₄₀R₄₁)_(p″)Y″(CR₂₄R₂₅)_(q)(CO)_(t)X″,—NR₃₃(C═O)_(p′)(CR₂₀R₂₁)_(m)A″_(m″)(CR₂₂R₂₃)_(n)(OCH₂CH₂)_(p)(CR₄₀R₄₁)_(p″)Y″(CR₂₄R₂₅)_(q)(CO)_(t)X″,—(CR₂₀R₂₁)_(m)(CR₂₂R₂₃)_(n)(OCH₂CH₂)_(p)(CR₄₀R₄₁)_(p″)Y″(CR₂₄R₂₅)_(q)(CO)_(t)X″,—(CR₂₀R₂₁)_(m)(CR₂₆═CR₂₇)_(m′)(CR₂₂R₂₃)_(n)(OCH₂CH₂)_(p)(CR₄₀R₄₁)_(p″)Y″(CR₂₄R₂₅)_(q)(CO)_(t)X″,—(CR₂₀R₂₁)_(m)(alkynyl)_(n′)(CR₂₂R₂₃)_(n)(OCH₂CH₂)_(p)(CR₄₀R₄₁)_(p″)Y″(CR₂₄R₂₅)_(q)(CO)_(t)X″,—(CR₂₀R₂₁)_(m)(piperazino)_(t′)(CR₂₂R₂₃)_(n)(OCH₂CH₂)_(p)(CR₄₀R₄₁)_(p″)Y″(CR₂₄R₂₅)_(q)(CO)_(t)X″,—(CR₂₀R₂₁)_(m)A″_(m″)(CR₂₂R₂₃)_(n)(OCH₂CH₂)_(p)(CR₄₀R₄₁)_(p″)Y″(CR₂₄R₂₅)_(q)(CO)_(t)X″,—(CR₂₀R₂₁)_(m)(CR₂₉═N—NR₃₀)_(n″)(CR₂₂R₂₃)_(n′)(OCH₂CH₂)_(p)(CR₄₀R₄₁)_(p″)Y″(CR₂₄R₂₅)_(q)(CO)_(t)X″,—(CR₂₀R₂₁)_(m)(CR₂₉═N—NR₃₀)_(n″)(CR₂₆═CR₂₇)_(m′)(CR₂₂R₂₃)_(n)(OCH₂CH₂)_(p)(CR₄₀R₄₁)_(p″)Y″(CR₂₄R₂₅)_(q)(CO)_(t)X″,—(CR₂₀R₂₁)_(m)(CR₂₉═N—NR₃₀)_(n″)(alkynyl)_(n′)(CR₂₂R₂₃)_(n)(OCH₂CH₂)_(p)(CR₄₀R₄₁)_(p″)Y″(CR₂₄R₂₅)_(q)(CO)_(t)X″,—(CR₂₀R₂₁)_(m)(CR₂₉═N—NR₃₀)_(n″)A″_(m″)(CR₂₂R₂₃)_(n)(OCH₂CH₂)_(p)(CR₄₀R₄₁)_(p″)Y″(CR₂₄R₂₅)_(q)(CO)_(t)X″,

wherein:

m, n, p, q, m′, n′, t′ are integer from 1 to 10, or are optionally 0;

t, m″, n″, and p″ are 0 or 1;

X″ is selected from OR₃₆, SR₃₇, NR₃₈R₃₉, wherein R₃₆, R₃₇, R₃₈, R₃₉ areH, or linear, branched or cyclic alkyl, alkenyl or alkynyl having from 1to 20 carbon atoms and, or, a polyethylene glycol unit —(OCH₂CH₂)_(n),R₃₇, optionally, is a thiol protecting group when t=1, COX″ forms areactive ester selected from N-hydroxysuccinimide esters,N-hydroxyphthalimide esters, N-hydroxy sulfo-succinimide esters,para-nitrophenyl esters, dinitrophenyl esters, pentafluorophenyl estersand their derivatives, wherein said derivatives facilitate amide bondformation;

Y″ is absent or is selected from O, S, S—S or NR₃₂, wherein R₃₂ has thesame definition as given above for R; or

when Y″ is not S—S and t=0, X″ is selected from a maleimido group, ahaloacetyl group or SR₃₇, wherein R₃₇ has the same definition as above;

A″ is an amino acid selected from glycine, alanine, leucine, valine,lysine, citrulline and glutamate or a polypeptide containing between 2to 20 amino acid units;

R₂₀, R₂₁, R₂₂, R₂₃, R₂₄, R₂₅, R₂₆, and R₂₇ are the same or different,and are —H or a linear or branched alkyl having from 1 to 5 carbonatoms;

R₂₉ and R₃₀ are the same or different, and are —H or alkyl from 1 to 5carbon atoms;

R₃₃ is —H or linear, branched or cyclic alkyl, alkenyl or alkynyl havingfrom 1 to 12 carbon atoms, a polyethylene glycol unit R—(OCH₂CH₂)_(n)—,or R₃₃ is —COR₃₄, —CSR₃₄, —SOR₃₄, or —SO₂R₃₄, wherein R₃₄ is H orlinear, branched or cyclic alkyl, alkenyl or alkynyl having from 1 to 20carbon atoms or, a polyethylene glycol unit —(OCH₂CH₂)_(n); and

one of R₄₀ and R₄₁ is optionally a negatively or positively chargedfunctional group and the other is H or alkyl, alkenyl, alkynyl having 1to 4 carbon atoms.

Any of the above linking groups may be present in any of the compounds,drug-linker compounds, or conjugates of the invention, includingreplacing the linking groups of any of the formulas described herein.

The term “amino acid” refers to naturally occurring amino acids ornon-naturally occurring amino acid represented byNH₂—C(R^(aa′)R^(aa))—C(═O)OH, wherein R^(aa) and R^(aa′) are eachindependently H, an optionally substituted linear, branched or cyclicalkyl, alkenyl or alkynyl having 1 to 10 carbon atoms, aryl, heteroarylor heterocyclyl. The term “amino acid” also refers to the correspondingresidue when one hydrogen atom is removed from the amine and/or carboxyend of the amino acid, such as —NH—C(R^(aa′)R^(aa))—C(═O)O—.

The term “cation” refers to an ion with positive charge. The cation canbe monovalent (e.g., Na⁺, K⁺, etc.), bi-valent (e.g., Ca²⁺, Mg²⁺, etc.)or multi-valent (e.g., Al³⁺ etc.). Preferably, the cation is monovalent.

The term “therapeutically effective amount” means that amount of activecompound or conjugate that elicits the desired biological response in asubject. Such response includes alleviation of the symptoms of thedisease or disorder being treated, prevention, inhibition or a delay inthe recurrence of symptom of the disease or of the disease itself, anincrease in the longevity of the subject compared with the absence ofthe treatment, or prevention, inhibition or delay in the progression ofsymptom of the disease or of the disease itself. Determination of theeffective amount is well within the capability of those skilled in theart, especially in light of the detailed disclosure provided herein.Toxicity and therapeutic efficacy of compound I can be determined bystandard pharmaceutical procedures in cell cultures and in experimentalanimals. The effective amount of compound or conjugate of the presentinvention or other therapeutic agent to be administered to a subjectwill depend on the stage, category and status of the multiple myelomaand characteristics of the subject, such as general health, age, sex,body weight and drug tolerance. The effective amount of compound orconjugate of the present invention or other therapeutic agent to beadministered will also depend on administration route and dosage form.Dosage amount and interval can be adjusted individually to provideplasma levels of the active compound that are sufficient to maintaindesired therapeutic effects.

Cytotoxic Compounds

The present invention is directed to cytotoxic compounds describedherein (e.g., compounds of formulas (I), (II), (III), and (IV)). In oneembodiment, the cytotoxic compounds of the present invention do notinclude any compounds described in US 2010/0203007 (the entire teachingof which is incorporated herein by reference), such as thosespecifically disclaimed in the proviso below.

In a first specific embodiment, the invention provides a cytotoxiccompound comprising a linking group with a reactive group bonded theretocapable of covalently linking the cytotoxic compound to a cell bindingagent (CBA), wherein said cytotoxic compound is represented by any oneof the following formulas (I), (II), (III) or (IV):

or a pharmaceutically acceptable salt thereof, wherein:

-   -   the double line        between N and C represents a single bond or a double bond,        provided that when it is a double bond, X is absent and Y is —H,        or a linear or branched alkyl having 1 to 4 carbon atoms, and        when it is a single bond, X is —H, the linking group with the        reactive group bonded thereto, or an amine protecting moiety;        preferably, the double line        between N and C represents a double bond;    -   Y is —H or a leaving group selected from —OR, —OCOR′, —OCOOR′,        —OCONR′R″, —NR′R″, —NR′COR″, —NR′NR′R″, an optionally        substituted 5- or 6-membered nitrogen-containing heterocycle        (e.g., piperidine, tetrahydropyrrole, pyrazole, morpholine,        etc.), a guanidinum represented by —NR′(C═NH)NR′R″, an amino        acid, or a peptide represented by —NRCOP′, wherein P′ is an        amino acid or a polypeptide containing between 2 to 20 amino        acid units, —SR, —SOR′, —SO₂M, —SO₃M, —OSO₃M, halogen, cyano and        an azido, wherein M is —H or a cation; such as Na⁺ or K⁺.        Preferably, M is —H or Na⁺. Preferably, Y is selected from        —SO₃M, —OH, —OMe, —OEt or —NHOH. More preferably, Y is —SO₃M or        —OH; or,    -   Y is a sulfite (HSO₃, HSO₂ or a salt of HSO₃ ⁻, SO₃ ²⁻or HSO₂ ⁻        formed with a cation), metabisulfite (H₂S₂O₅ or a salt of S₂O₅        ²⁻ formed with a cation), mono-, di-, tri-, and        tetra-thiophosphate (PO₃SH₃, PO₂S₂H₂, POS₃H₂, PS₄H₂ or a salt of        PO₃S³⁻, PO₂S₂ ³⁻, POS₃ ⁻ or PS₄ ³⁻ formed with a cation), thio        phosphate ester (R^(i)O)₂PS(OR^(i)), R^(i)S—, R^(i)SO, R^(i)SO₂,        R^(i)SO₃, thiosulfate (HS₂O₃ or a salt of S₂O₃ ²⁻ formed with a        cation), dithionite (HS₂O₄ or a salt of S₂O₄ ²⁻ formed with a        cation), phosphorodithioate (P(═S)(OR^(k′))(S)(OH) or a salt        thereof formed with a cation), hydroxamic acid (R^(k′)C(═O)NOH        or a salt formed with a cation), formaldehyde sulfoxylate        (HOCH₂SO₂ ⁻ or a salt of HOCH₂SO₂ ⁻ formed with a cation, such        as HOCH₂SO₂ ⁻Na⁺) or a mixture thereof, wherein R^(i) is a        linear or branched alkyl having 1 to 10 carbon atoms and is        substituted with at least one substituent selected from        —N(R^(j))₂, —CO₂H, —SO₃H, and —PO₃H; R^(i) can be further        optionally substituted with a substituent for an alkyl described        herein; R^(j) is a linear or branched alkyl having 1 to 6 carbon        atoms; R^(k′) is a linear, branched or cyclic alkyl, alkenyl or        alkynyl having 1 to 10 carbon atoms, aryl, heterocyclyl or        heteroaryl; preferably, Y is an adduct of a bisulfite, a        hydrosulfite, or a metabisulfite, or salts thereof (such as        sodium salt);    -   R, for each occurrence, is independently selected from the group        consisting of —H, an optionally substituted linear, branched or        cyclic alkyl, alkenyl or alkynyl having from 1 to 10 carbon        atoms, a polyethylene glycol unit —(CH₂CH₂O)_(n)—R^(c), an        optionally substituted aryl having 6 to 18 carbon atoms, an        optionally substituted 5- to 18-membered heteroaryl ring        containing one or more heteroatoms independently selected from        nitrogen, oxygen, and sulfur, or an optionally substituted 3- to        18-membered heterocyclic ring containing 1 to 6 heteroatoms        independently selected from O, S, N and P;    -   R′ and R″ are the same or different, and are independently        selected from —H, —OH, —OR, —NHR, —NR₂, —COR, an optionally        substituted linear, branched or cyclic alkyl, alkenyl or alkynyl        having from 1 to 10 carbon atoms, a polyethylene glycol unit        —(CH₂CH₂O)_(n)—R^(c), and an optionally substituted 3- to        18-membered heterocyclic ring having 1 to 6 heteroatoms        independently selected from O, S, N and P;    -   R^(c) is —H or a substituted or unsubstituted linear or branched        alkyl having 1 to 4 carbon atoms, or the linking group with the        reactive group bonded thereto;    -   n is an integer from 1 to 24;    -   W is selected from C═O, C═S, CH₂, BH, SO, and SO₂;    -   X′ is selected from the group consisting of —H, —OH, an        amine-protecting group, the linking group with the reactive        group bonded thereto, an optionally substituted linear, branched        or cyclic alkyl, alkenyl or alkynyl having from 1 to 10 carbon        atoms, a polyethylene glycol unit —(CH₂CH₂O)_(n)—R^(c), an        optionally substituted aryl having 6 to 18 carbon atoms (e.g.,        phenyl), an optionally substituted 5- to 18-membered heteroaryl        ring containing one or more heteroatoms independently selected        from nitrogen, oxygen, and sulfur, and an optionally substituted        3- to 18-membered heterocyclic ring containing 1 to 6        heteroatoms independently selected from O, S, N and P.        Preferably, X′ is —H, —OH, -Me or the linking group with the        reactive group bonded thereto. More preferably, X′ is —H;    -   Y′ is selected from the group consisting of —H, an oxo group,        the linking group with the reactive group bonded thereto, an        optionally substituted linear, branched or cyclic alkyl, alkenyl        or alkynyl having from 1 to 10 carbon atoms, an optionally        substituted 6- to 18-membered aryl, an optionally substituted 5-        to 18-membered heteroaryl ring containing one or more        heteroatoms independently selected from nitrogen, oxygen, and        sulfur, an optionally substituted 3- to 18-membered heterocyclic        ring having 1 to 6 heteroatoms. Preferably, Y′ is selected from        —H or oxo. More preferably, Y′ is —H;    -   R₁, R₂, R₃, R₄, R₁′, R₂′, R₃′ and R₄′ are each independently        selected from the group consisting of —H, an optionally        substituted linear, branched or cyclic alkyl, alkenyl or alkynyl        having from 1 to 10 carbon atoms, a polyethylene glycol unit        —(OCH₂CH₂)_(n)—R^(c), halogen, guanidinium [—NH(C═NH)NH₂], —OR,        —NR′R″, —NO₂, —NCO, —NR′COR″, —SR, a sulfoxide represented by        —SOR′, a sulfone represented by —SO₂R′, a sulfonate —SO₃ ⁻M⁺, a        sulfate —OSO₃ ⁻M⁺, a sulfonamide represented by —SO₂NR′R″,        cyano, an azido, —COR′, —OCOR′, —OCONR′R″ and the linking group        with the reactive group bonded thereto. Preferably, one of R₂,        R₃, R₂′ and R₃′ is the linking group with the reactive group        bonded thereto and the rest are —H;    -   R₆ is —H, —R, —OR, —SR, —NR′R″, —NO₂, halogen, the linking group        with the reactive group bonded thereto, —OR′ or —SR′, wherein        R^(c) is —H, a linear or branched alkyl having 1 to 4 carbon        atoms. Preferably, R₆ is —OMe or —SMe. Even more preferably, R₆        is —OMe;    -   Z and Z′ are independently selected from —(CH₂)_(n′)—,        —(CH₂)_(n′)—CR₇R₈—(CH₂)_(na′)—, —(CH₂)_(n′)—NR₉—(CH₂)_(na′)—,        —(CH₂)_(n′)—O—(CH₂)_(na′)— and —(CH₂)_(n′)—S—(CH₂)_(na′)—;    -   n′ and na′ are same or different, and are selected from 0, 1, 2        and 3;    -   R₇ and R₈ are the same or different, and are each independently        selected from —H, —OH, —SH, —COOH, —NHR′, a polyethylene glycol        unit —(OCH₂CH₂)_(n)—, an amino acid, a peptide unit bearing 2 to        6 amino acids, an optionally substituted linear, branched or        cyclic alkyl having from 1 to 10 carbon atoms;    -   R₉ is independently selected from —H, an optionally substituted        linear, branched or cyclic alkyl having from 1 to 10 carbon        atoms, a polyethylene glycol unit —(OCH₂CH₂)_(n)—;    -   A and A′ are the same or different, and are independently        selected from —O—, oxo (—C(═O)—), —CRR′O—, —CRR′—, —S—, —CRR′S—,        —N(R₅)— and —CRR′N(R₅)—. Preferably, A and A′ are the same or        different, and are selected from —O— and —S—. More preferably, A        and A′ are —O—;    -   R₅ for each occurrence is independently —H or an optionally        substituted linear or branched alkyl having 1 to 10 carbon        atoms;    -   D and D′ are the same or different, and are independently absent        or selected from the group consisting of an amino acid, a        peptide bearing 2 to 6 amino acids, and a polyethylene glycol        unit (—OCH₂CH₂)_(n)—, an optionally substituted linear, branched        or cyclic alkyl, alkenyl or alkynyl having 1 to 10 carbon atoms,        wherein the alkyl, alkenyl and alkynyl are optionally        substituted with one or more (e.g., 2, 3, 4, 5, 6 or more)        substituents independently selected from the group consisting of        halogen, —OR, —NR′COR″, —SR and —COR′;    -   Preferably, D and D′ are the same or different, and are        independently selected from linear, branched or cyclic alkyl,        alkenyl or alkynyl having from 1 to 10 carbon atoms. More        preferably, D and D′ are linear or branched alkyl bearing 1 to 4        carbon atoms. Still more preferably, D and D′ are the same or        different, and are selected from a linear alkyl having 1 to 4        carbon atoms;    -   L is absent, the linking group with the reactive group bonded        thereto, a polyethylene glycol unit (—OCH₂CH₂)_(n)—, a linear,        branched or cyclic alkyl or alkenyl having 1 to 10 carbon atoms        (e.g., 1-6 carbon atoms), a phenyl group, a 3- to 18-membered        heterocyclic ring or a 5- to 18-membered heteroaryl ring having        1 to 6 heteroatoms independently selected from O, S, N and P,        wherein the alkyl or alkenyl is optionally substituted with the        linking group with the reactive group bonded thereto; phenyl or        heterocyclic or heteroaryl ring can be optionally substituted,        wherein the substituent can be the linking group with the        reactive group bonded thereto.

In certain embodiments, X is not the linking group with the reactivegroup bonded thereto. In certain embodiments, the double line

between N and C represents a single bond, Y is not —H.

In certain embodiments, the cytotoxic compounds of the present inventionare not any one of the following compounds:

In certain embodiments, Y is —H or a leaving group selected from —OR,—OCOR′, —OCOOR′, —OCONR′R″, —NR′R″, —NR′COR″, —NR′NR′R″, an optionallysubstituted 5 or 6-membered nitrogen-containing heterocycle (e.g.,piperidine, tetrahydropyrrole, pyrazole, morpholine, etc.), a guanidinumrepresented by —NR′(C═NH)NR′R″, an amino acid, or a peptide representedby —NRCOP′, wherein P′ is an amino acid or a polypeptide containingbetween 2 to 20 amino acid units, —SR, —SOR′, —SO₂M, —SO₃M, —OSO₃M,halogen, cyano and an azido. Preferably, Y is Sodium Bisulfite adduct,Sodium Hydrosulfite adduct, or Sodium Metabisulfite adduct. In certainembodiments, Y is not —H.

In certain embodiments, L is absent, or is selected from an optionallysubstituted phenyl group and an optionally substituted pyridyl group,wherein the phenyl and the pyridyl group bears the linking group withthe reactive group bonded thereto, or L is an amine group bearing thelinking group with the reactive group bonded thereto (i.e., —N(linkinggroup)-), or L is a linear, branched or cyclic alkyl or alkenyl havingfrom 1 to 6 carbon atoms and bearing the linking group with the reactivegroup bonded thereto.

In a second specific embodiment, for cytotoxic dimers (I), (II), (III)and (IV), the variables are as described below:

the double line

between N and C represents a double bond;

Y is —H;

W is C═O;

R₁, R₂, R₁′, R₂′, R₄ and R₄′ are —H;

one of R₃, or R₃′ is optionally a linking group and the other is —H;

R₆ is —OMe;

Z and Z′ are —CH₂—;

X′ is —H;

Y′ is —H;

A and A′ are —O—; and the remainder of the variables are as described inthe first specific embodiment.

In a third specific embodiment, the cytotoxic dimers of formula (I),(II), (III) and (IV) are represented by the following formulas:

wherein:

-   -   the double line        between N and C represents a single bond or a double bond,        provided that when it is a double bond X is absent and Y is —H,        and when it is a single bond, X is selected from —H, the linking        group with the reactive group bonded thereto, or an amine        protecting group (preferably X is —H);    -   Y is selected from —H, —OR, —OCOR′, —SR, —NR′R″, —SO₃M, —SO₂M,        or —OSO₃M, wherein M is —H or a cation such as Na⁺. K⁺.        Preferably, Y is selected from —OH, —OMe, —OEt, —NHOH or —SO₃M.        Even more preferably, Y is —OH or —SO₃M, preferably M is —H or        Na⁺;    -   R is —H, an optionally substituted linear, branched or cyclic        alkyl, alkenyl or alkynyl having from 1 to 10 carbon atoms or a        PEG group —(CH₂CH₂O)_(n)—R^(c), wherein n is an integer from 1        to 24 and R^(c) is a linear or branched alkyl having 1 to 4        carbon atoms;    -   R′ and R″ are the same or different, and are selected from —H,        —OH, —OR, —NRR^(g′), —COR, an optionally substituted linear,        branched or cyclic alkyl, alkenyl or alkynyl having from 1 to 10        carbon atoms, an optionally substituted aryl having from 6 to 18        carbon atoms, an optionally substituted 3- to 18-membered        heterocyclic ring having 1 to 6 heteroatoms selected from O, S,        N and P, a PEG group —(CH₂CH₂O)_(n)—R^(c), wherein n is an        integer from 1 to 24, preferably n is 2, 4 or 8; and R^(g′) is        —H, an optionally substituted linear, branched or cyclic alkyl,        alkenyl or alkynyl having from 1 to 10 carbon atoms or a PEG        group —(CH₂CH₂O)_(n)—R^(c);    -   X′ is selected from the group consisting of —H, —OH, a        substituted or unsubstituted linear, branched or cyclic alkyl,        alkenyl or alkynyl having from 1 to 10 carbon atoms, phenyl, and        an amine-protecting group. Preferably, X′ is —H, —OH or -Me.        More preferably, X′ is —H;    -   Y′ is selected from the group consisting of —H, an oxo group, a        substituted or unsubstituted linear, branched or cyclic alkyl,        alkenyl or alkynyl having from 1 to 10 carbon atoms. Preferably,        Y′ is selected from —H or -Me. More preferably Y′ is —H;    -   R₆ is —OR^(c) or —SR^(c), wherein R^(c) is a linear or branched        alkyl having 1 to 4 carbon atoms. Preferably, R₆ is —OMe or        —SMe. Even more preferably, R₆ is —OMe;    -   A and A′ are selected from —O— and —S—. Preferably, A and A′ are        —O—;    -   L′, L″, and L′″ are the same or different, and are independently        selected from —H, an optionally substituted linear, branched or        cyclic alkyl, alkenyl or alkynyl having from 1 to 10 carbon        atoms, a polyethylene glycol unit —(OCH₂CH₂)_(n)—R^(c), halogen,        guanidinium [—NH(C═NH)NH₂], —OR, —NR′R″, —NO₂, —NR′COR″, —SR, a        sulfoxide represented by —SOR′, a sulfone represented by —SO₂R′,        a sulfonate —SO₃ ⁻M⁺, a sulfate —OSO₃ ⁺M⁻, a sulfonamide        represented by SO₂NR′R″, cyano, an azido, —COR′, —OCOR′,        —OCONR′R″ and the linking group with the reactive group bonded        thereto, provided only one of L′, L″, and L′″ is the linking        group with the reactive group bonded thereto. Preferably, L′ is        the linking group with the reactive group bonded thereto.        Alternatively, one of L′, L″ or L′″ is the linking group with        the reactive group bonded thereto, while the others are —H. More        preferably, L′ is the linking group with the reactive group        bonded thereto, and L″ and L′″ are —H;    -   G is selected from —CH— or —N—; and the remainder of the        variables are as described in the first specific embodiment.

In certain embodiments, X is not the linking group with the reactivegroup bonded thereto. In certain embodiments, the double line

between N and C represents a single bond, Y is not —H.

In certain embodiments, A and A′ are both —O—, R₆ is —OMe, and G is—CH—.

In a fourth specific embodiment, for the cytotoxic dimers of formula(IA), (IIA), (IIIA) or (IVA), L′ is represented by the formula:

—W′—R^(x)—V—R^(y)-J,

wherein:

-   -   W′ and V are the same or different, and are each independently        absent, or selected from —CR^(e)R^(e′)—, —O—, —O—C(═O)—,        —C(═O)—O—, —S—, —SO—, —SO₂—, —CH₂—S—, —CH₂O—, —CH₂NR^(e)—,        —O—(C═O)O—, —O—(C═O)N(R^(e))—, —N(R^(e))—C(═O)—,        —C(═O)—N(R^(e))—, —N(R^(e))—C(═O)O—, —N(C(═O)R^(e))C(═O)—,        —N(C(═O)R^(e))—, —(O—CH₂—CH₂)_(n)—, —SS—, or —C(═O)—, or an        amino acid, or a peptide having 2 to 8 amino acids;    -   R^(x) and R^(y) are the same or different, and are each        independently absent or an optionally substituted linear,        branched or cyclic alkyl, alkenyl or alkynyl having 1 to 10        carbon atoms, an aryl bearing 6 to 10 carbon atoms or a 3- to        8-membered hetereocyclic ring bearing 1 to 3 heteroatoms        selected from O, N or S;    -   R^(e) and R^(e′) are the same or different, and are selected        from —H, a linear, branched or cyclic alkyl, alkenyl or alkynyl        having 1 to 10 carbon atoms or —(CH₂—CH₂—O)_(n)—R^(k), wherein        R^(k) is a —H, a linear, branched cyclic alkyl having 1 to 6        carbon atoms, optionally bearing a secondary amino (e.g.,        —NHR¹⁰¹) or tertiary amino (—NR¹⁰¹R¹⁰²) group or a 5 or        6-membered nitrogen containing heterocycle, such as piperidine        or morpholine, wherein R¹⁰¹ and R¹⁰² are each independently a        linear, branched, or cyclic alkyl, alkenyl or alkynyl having 1        to 10 carbon atoms. Preferably, R¹⁰¹ and R¹⁰² are each        independently a linear or branched alkyl having 1 to 6 carbon        atoms;    -   n is an integer from 1 to 24; and    -   J comprises the reactive group bonded thereto, and is selected        from a maleimide, a haloacetamido, —SH, —SSR^(d), —CH₂SH,        —CH(Me)SH, —C(Me)₂SH, —NHR^(c1), —CH₂NHR^(c1), —NR^(c1)NH₂,        —COOH, and —COE, wherein —COE represents a reactive ester        selected from, but not limited to, N-hydroxysuccinimde ester,        N-hydroxy sulfosuccinimide ester, nitrophenyl (e.g., 2 or        4-nitrophenyl) ester, dinitrophenyl (e.g., 2,4-dinitrophenyl)        ester, sulfo-tetrafluorophenyl (e.g.,        4-sulfo-2,3,5,6-tetrafluorophenyl) ester, and pentafluorophenyl        ester, and wherein R^(c1) is —H or a substituted or        unsubstituted linear or branched alkyl having 1 to 4 carbon        atoms, and    -   R^(d) is selected from phenyl, nitrophenyl (e.g., 2 or        4-nitrophenyl), dinitrophenyl (e.g., 2 or 4-nitrophenyl),        carboxynitrophenyl (e.g., 3-carboxy-4-nitrophenyl), pyridyl or        nitropyridyl (e.g., 4-nitropyridyl).

In certain embodiments, J is —SH, —SSR^(d), a maleimide, or aN-hydroxysuccinimide ester.

In certain embodiments, R^(e′) is —H or -Me; R^(e) is a linear orbranched alkyl having 1 to 6 carbon atoms or —(CH₂—CH₂—O)_(n)—R^(k); nis an integer from 2 to 8; preferably R^(k) is —H, -Me or —CH₂CH₂—NMe₂,and the remainder of the variables are as described above in the fourthspecific embodiment.

In certain embodiments, V is an amino acid or a peptide having 2 to 8amino acids. In certain embodiments, V is valine-citrulline,gly-gly-gly, or ala-leu-ala-leu.

In certain embodiments,

-   -   W′ is —O—, —N(R^(e))— or —N(R^(e))—C(═O)—;    -   R^(e) is H, a linear or branched alkyl having 1 to 4 carbon        atoms, or —(CH₂—CH₂—O)_(n)—R^(k);    -   R^(x) is a linear or branched alkyl having 1 to 6 carbon atoms;    -   V is absent, —(O—CH₂—CH₂)_(n)—, —C(═O)—NH—, —S—, —NH—C(═O)—;    -   R^(y) is absent or a linear or branched alkyl having 1 to 4        carbon atoms; and    -   J is —SH, —SSR^(d) or —COE (preferably, N-hydroxysuccinimide        ester). The remainder of the variables is as described in the        fourth specific embodiment.

In certain embodiments,

-   -   W′ is —O—, —N(R^(e))— or —N(R^(e))—C(═O)—;    -   R^(e) is H, Me, or —(CH₂—CH₂—O)_(n)-Me;    -   n is an integer from 2 to 6;    -   R^(x) is linear or branched alkyl bearing 1 to 6 carbon atoms;    -   V and R^(y) are absent; and    -   J is —COE, preferably N-hydroxysuccinimide ester. The remainder        of the variables is as described in the fourth specific        embodiment.

In a fifth specific embodiment, L′ is represented by the followingformula:

—W′-[CR_(1″)R_(2″)]_(a)—V—[Cy]₀₋₁-[CR_(3″)R_(4″)]_(b)—COE,

wherein:

-   -   R_(1″), R_(2″), and R_(3″) are each independently —H or a linear        or branched alkyl bearing 1 to 4 carbon atoms, preferably -Me;    -   R^(4″) is —H, a linear or branched alkyl bearing 1 to 4 carbon        atoms (preferably -Me), —SO₃H, or —SO₃ ⁻M⁺, wherein M⁺ is a        pharmaceutically acceptable cation;    -   a is an integers from 0-5 (e.g., from 0 to 2, 3, 4, or 5), and b        is an integer from 0-6 (e.g., from 0 to 3, 4, 5, or 6); and,    -   Cy is an optionally substituted 5-membered heterocyclic ring        bearing an N heteroatom, preferably Cy is

In certain embodiments, W′ is —N(R^(e))—.

In certain embodiments, such as in the fourth and/or the fifth specificembodiment, R^(e) is —(CH₂—CH₂—O)₂₋₆—R^(k), wherein R^(k) is a —H, alinear, branched cyclic alkyl having 1 to 6 carbon atoms.

In certain embodiments, such as in the fourth and/or the fifth specificembodiment, V is —S— or —SS—.

In a sixth specific embodiments, such as in the fourth and/or the fifthspecific embodiment, L′ is represented by the following formula:

—NR^(e)—[CR_(1″)R_(2″)]_(a)—S—[CR_(3″)R_(4″)]_(b)—COE.

In certain embodiments, the compound is any of the following:

-   -   wherein Y is —H or —SO₃M, and M is —H or a pharmaceutically        acceptable cation. In certain embodiments, Y is —SO₃M.

In a seventh specific embodiments, such as in the fourth and/or thefifth specific embodiment, L′ is represented by the following formula:

—NR^(e)—[CR_(1″)R_(2″)]_(a)—S—Cy-[CR_(3″)R_(4″)]_(b)—COE.

In certain embodiments, the compound is any one of the following:

-   -   wherein Y is —H or —SO₃M, and M is —H or a pharmaceutically        acceptable cation. In certain embodiments, Y is —SO₃M.

In a eighth specific embodiment, the cytotoxic dimers of formula (I),(II), (III) and (IV) are represented by the following formulas:

wherein:

-   -   W′ is absent, or selected from —O—, —N(R^(e))—,        —N(R^(e))—C(═O)—, —N(C(═O)R^(e))—, —S— or —CH₂—S—, —CH₂NR^(e)—;    -   R^(x) is absent or selected from a linear, branched or cyclic        alkyl having 1 to 10 carbon atoms;    -   R^(e) is —H, a linear, branched or cyclic alkyl, alkenyl or        alkynyl having 1 to 10 carbon atoms or —(CH₂—CH₂—O)_(n)—R^(k),        wherein R^(k) is a —H, a linear, branched cyclic alkyl having 1        to 6 carbon atoms, optionally bearing a secondary amino (e.g.,        —NHR¹⁰¹) or tertiary amino (—NR¹⁰¹R¹⁰²) group or a 5- or        6-membered nitrogen containing heterocycle, such as piperidine        or morpholine, wherein 8101 and R¹⁰² are each independently a        linear, branched, or cyclic alkyl, alkenyl or alkynyl having 1        to 10 carbon atoms. Preferably, R¹⁰¹ and R^(m2) are each        independently a linear or branched alkyl having 1 to 6 carbon        atoms;    -   Z^(s) is —H, —SR^(m);    -   R^(m) is R^(d) or a substituted or unsubstituted linear or        branched alkyl having 1 to 4 carbon atoms bearing a reactive        ester, selected from N-hydroxysuccinimide esters,        N-hydroxyphtalimide esters, N-hydroxy sulfo-succinimide esters,        para-nitrophenyl esters, dinitrophenyl esters, pentafluorophenyl        esters;    -   R^(d) is selected from phenyl, nitrophenyl, dinitrophenyl,        carboxynitrophenyl, pyridyl or nitropyridyl; and,    -   n is an integer from 1 to 24; and the remainder of the variables        are as described above in the third specific embodiment.

Preferably, R^(k) is —H or -Me, and n is an integer from 2 to 8.Preferably, R^(x) is a linear or branched alkyl having 1 to 6 carbonatoms; and the remainder of the variables is as described above in thethird, fourth, and/or the fifth specific embodiment.

In a ninth specific embodiment, the cytotoxic dimers of formula (I),(II), (III) and (IV) are represented by the following formulas:

wherein:

-   -   the double line        between N and C represents a single bond or a double bond,        provided that when it is a double bond, X is absent and Y is —H,        and when it is a single bond, X is selected from —H, the linking        group with the reactive group bonded thereto, or an amine        protecting group (preferably X is —H or an amine protecting        group; more preferably, X is —H);    -   Y is selected from —H, —OR, —OCOR′, —SR, —NR′R,″ —SO₃M, —SO₂M or        —OSO₃M (e.g., Y is —OR, —OCOR′, —SR, —NR′R,′ —SO₃M, —SO₂M or        —OSO₃M), wherein M is —H or a cation such as Na⁺ or K⁺;    -   R is —H, an optionally substituted linear, branched or cyclic        alkyl, alkenyl or alkynyl having from 1 to 10 carbon atoms or a        PEG group —(CH₂CH₂O)_(n)—R^(c), wherein n is an integer from 1        to 24, and R^(c) is a linear or branched alkyl having 1 to 4        carbon atoms;    -   R′ and R″ are the same or different, and are selected from —H,        —OH, —OR, —NRR^(g′), —COR, an optionally substituted linear,        branched or cyclic alkyl, alkenyl or alkynyl having from 1 to 10        carbon atoms, an optionally substituted aryl having from 6 to 18        carbon atoms, an optionally substituted 3- to 18-membered        heterocyclic ring having 1 to 6 heteroatoms selected from O, S,        N and P, a PEG group —(CH₂CH₂O)_(n)—R^(c), wherein n is an        integer from 1 to 24, preferably n is 2, 4 or 8; and R^(g′) is        —H, an optionally substituted linear, branched or cyclic alkyl,        alkenyl or alkynyl having from 1 to 10 carbon atoms or a PEG        group —(CH₂CH₂O)_(n)—R^(c);    -   X′ is selected from the group consisting of —H, —OH, a        substituted or unsubstituted linear, branched or cyclic alkyl,        alkenyl or alkynyl having from 1 to 10 carbon atoms, phenyl, and        an amine-protecting group;    -   Y′ is selected from the group consisting of —H, an oxo group, a        substituted or unsubstituted linear, branched or cyclic alkyl,        alkenyl or alkynyl having from 1 to 10 carbon atoms;    -   A and A′ are selected from —O— and —S—;    -   W′ is absent, or selected from —O—, —N(R^(e))—,        —N(R^(e))—C(═O)—, —N(C(═O)R^(e))—, —S— or —CH₂—S—, —CH₂NR^(e)—;    -   R^(x) is absent or selected from a linear, branched or cyclic        alkyl having 1 to 10 carbon atoms;    -   R^(e) is —H, a linear, branched or cyclic alkyl, alkenyl or        alkynyl having 1 to 10 carbon atoms or —(CH₂—CH₂—O)_(n)—R^(k),        wherein R^(k) is a —H, a linear, branched cyclic alkyl having 1        to 6 carbon atoms, optionally bearing a secondary amino (e.g.,        —NHR¹⁰¹) or tertiary amino (—NR¹⁰¹R¹⁰²) group or a 5 or        6-membered nitrogen containing heterocycle, such as piperidine        or morpholine, wherein R¹⁰¹ and R¹⁰² are each independently a        linear, branched, or cyclic alkyl, alkenyl or alkynyl having 1        to 10 carbon atoms;    -   G is selected from —CH— or —N—;    -   Z^(s) is —H, or is selected from any one of the following        formulas:

wherein:

-   -   q is an integer from 1 to 5;    -   n is an integer from 2 to 6;    -   D is —H or —SO₃M;    -   M is —H or a cation, such as Na⁺ or K⁺.

In certain embodiments, Z^(s) is represented by any one of the followingformulas:

In certain embodiments, W′ is —N(R^(e))—.

In certain embodiments, R^(e) is —(CH₂—CH₂—O)_(n)—R^(k), wherein R^(k)is a —H, a linear, branched cyclic alkyl having 1 to 6 carbon atoms.

In certain embodiments, R^(k) is —H or -Me, n is 4, and q is 2.

In certain embodiments, R^(x) is a linear or branched alkyl having 1 to6 carbon atoms.

In certain embodiments, R^(x) may be —(CH₂)_(p)—(CR^(f)R^(g))—, whereinR^(f) and R^(g) are each independently selected from H or a linear orbranched alkyl having 1 to 4 carbon atoms; and p is 0, 1, 2 or 3.

In certain embodiments, R^(f) and R^(g) are the same or different, andare selected from —H and -Me; and p is 1.

In a tenth specific embodiment, the compounds of formula (VIII), (IX),(X) and (XI) described in the ninth specific embodiment, the variablesare as described below:

-   -   the double line        between N and C represents a single bond or a double bond,        provided that when it is a double bond X is absent and Y is —H,        and when it is a single bond, X is —H; Y is —H, —OH or —SO₃M;    -   M is —H or a pharmaceutically acceptable cation (e.g., Na⁺);    -   X′ and Y′ are both —H;    -   A and A′ are both —O—;    -   R₆ is —OMe; and    -   R^(x) is a linear or branched alkyl having 1 to 6 carbon atoms.

In a related embodiment, Y is —OH or —SO₃M.

In another embodiment, the compounds of formula (VIII), (IX), (X) and(XI) described in the ninth specific embodiment, the variables are asdescribed below:

-   -   W′ is —O—, —N(R^(e))—, —N(R^(e))—C(═O)—, —N(COR^(e))—, —S— or        —CH₂—S—;    -   R^(x) is absent or selected from a linear, branched or cyclic        alkyl having 1 to 6 carbon atoms;    -   R^(e) is —H, a linear, branched or cyclic alkyl, alkenyl or        alkynyl having 1 to 10 carbon atoms or —(CH₂—CH₂—O)_(n)—R^(k),        wherein R^(k) is a —H, a linear, branched cyclic alkyl having 1        to 6 carbon atoms, optionally bearing a primary, secondary or        tertiary amino group or a 5- or 6-membered Nitrogen containing        heterocycle, such as piperidine or morpholine;    -   n is an integer from 1 to 24; and the remainder of the variables        are as described above in the ninth specific embodiment.

Preferably, R^(k) is —H or -Me, and n is an integer from 2 to 8.Preferably, R^(x) is a linear or branched alkyl having 1 to 6 carbonatoms.

Preferably, R^(x) is —(CH₂)_(p)—(CR^(f)R^(g))—, wherein R^(f) and R^(g)are each independently selected from H or a linear or branched alkylhaving 1 to 4 carbon atoms; p is 0, 1, 2 or 3. More preferably, R^(f)and R^(g) are the same or different, and are selected from —H and -Me;and p is 1.

In another preferred embodiment, the linker is represented by any one ofthe formula selected from formulas (a1), (a4), (a5), (a10) and (a11)shown above; and the remainder of the variables are as described abovein the tenth specific embodiment.

In a eleventh specific embodiment, for compounds of formula (IB), (IIB),(IIIB) and (IVB) described in the eighth specific embodiment, thevariables are as described below:

-   -   the double line        between N and C represents a single bond or a double bond,        provided that when it is a double bond X is absent and Y is —H,        and when it is a single bond, X is —H; Y is —H, —OH or —SO₃M        (e.g., Y is —OH or —SO₃M);    -   M is —H or Na⁺;    -   X′ and Y′ are both —H;    -   A and A′ are both —O—;    -   R₆ is —OMe;    -   R^(x) is a linear or branched alkyl having 1 to 6 carbon atoms;        and the remainder of the variables is as described above in the        third, fourth, or the fifth specific embodiment.

Preferably, R^(x) is —(CH₂)_(p)—(CR^(f)R^(g))—, wherein R^(f) and R^(g)are each independently selected from H or a linear or branched alkylhaving 1 to 4 carbon atoms; p is 0, 1, 2 or 3. More preferably, R^(f)and R^(g) are the same or different, and are selected from —H and -Me;and p is 1.

In any of the specific embodiments above (e.g., the first to the 11^(th)specific embodiments), the double line

between N and C may represent a double bond.

In any of the specific embodiments above (e.g., the first to the 11^(th)specific embodiments), the double line

between N and C may represent a single bond, X is —H, the linking groupwith the reactive group bonded thereto, or an amine protecting group(e.g., X is —H or an amine protecting group); and Y is selected from —H,—OR, —OCOR′, —SR, —NR′R,″ an optionally substituted 5- or 6-memberednitrogen-containing heterocycle, —SO₃M, —SO₂M and a sulfate —OSO₃M(e.g., Y is —OR, —OCOR′, —SR, —NR′R,″ an optionally substituted 5- or6-membered nitrogen-containing heterocycle, —SO₃M, —SO₂M and a sulfate—OSO₃M).

In certain embodiments, Y is selected from —H, —SO₃M, —OH, —OMe, —OEt or—NHOH (e.g., Y is —SO₃M, —OH, —OMe, —OEt or —NHOH).

In certain embodiments, Y is —H, —SO₃M or —OH (e.g., Y is —SO₃M or —OH).

In certain embodiments, M is —H, Na⁺ or K⁺.

In any of the specific embodiments above (e.g., the first to the 11^(th)specific embodiments), W, when present, is C═O.

In any of the specific embodiments above (e.g., the first to the 11^(th)specific embodiments), Z and Z′, when present, are —CH₂.

In any of the specific embodiments above (e.g., the first to the 11^(th)specific embodiments), X′ is selected from the group consisting of —H,—OH, an optionally substituted linear, branched or cyclic alkyl, alkenylor alkynyl having from 1 to 10 carbon atoms, phenyl, the linking groupwith the reactive group bounded thereto, and an amine-protecting group.

In certain embodiments, X′ is —H, —OH, -Me or the linking group with thereactive group bounded thereto.

In certain embodiments, X′ is —H.

In any of the specific embodiments above (e.g., the first to the 11^(th)specific embodiments), Y′ is selected from the group consisting of —H,an oxo group, a substituted or unsubstituted linear, branched or cyclicalkyl, alkenyl or alkynyl having from 1 to 10 carbon atoms.

In certain embodiments, Y′ is —H or oxo.

In certain embodiments, Y′ is —H.

In any of the specific embodiments above (e.g., the first to the 11^(th)specific embodiments), A and A′ are the same or different, and areselected from O, S, NR₅ and oxo (C═O). A and A′ may be same or differentand selected from —O— and —S—. Preferably, both A and A′ are —O—.

In any of the specific embodiments above (e.g., the first to the 11^(th)specific embodiments), D and D′, when present, are the same ordifferent, and are independently selected from a polyethylene glycolunit (—OCH₂CH₂)_(n), wherein n is an integer from 1 to 24, an aminoacid, a peptide bearing 2 to 6 amino acids, or a linear, branched orcyclic alkyl, alkenyl or alkynyl having 1 to 10 carbon atoms, whereinthe alkyl, alkenyl and alkynyl are optionally substituted with one ormore substituents independently selected from the group consisting ofhalogen, —OR, —NR′COR″, —SR and —COR′. Preferably, D and D′ are linearor branched alkyl bearing 1 to 4 carbon atoms.

In a twelfth embodiment, the cytotoxic compound of the present inventionas described in the first, third, and ninth embodiment is represented bythe following:

-   -   the double line        between N and C represents a double bond;    -   Y is —H;    -   W is C═O;    -   R₁, R₂, R₁′, R₂′, R₄ and R₄′ are —H;    -   one of R₃, or R₃′ is optionally the linking group with the        reactive group bounded thereto and the other is —H;    -   R₆ is —OMe;    -   Z and Z′ are —CH₂;    -   X′ is —H;    -   Y′ is —H; and    -   A and A′ are —O—.

In a thirteenth embodiment, the cytotoxic compound of the presentinvention is:

or a pharmaceutically acceptable salt thereof.

In one embodiment, compound 29b can used in methods of the presentinvention described herein. In a preferred embodiment, compound 29b canbe used for treating a proliferative disorder, such as cancer.

In another embodiment, compound 29b can be used for screening cell linesto identify cell lines that are sensitive to benzodiazepine compounds,such as benzodiazepine derivatives described herein.

Drug Compounds & Drug-Linker Compounds

The cytotoxic compounds described above comprise a linking group with areactive group bonded thereto, which compounds may result from reactinga bifunctional crosslinking reagent with “linker-less” compounds to formthe so-called drug-linker compounds. Alternatively, drug compounds thatare otherwise identical to the drug-linker compounds, but without thelinker moiety are also encompassed by the present invention.

Thus in certain embodiments, the invention provides a cytotoxic compoundwithout linking group, but may be capable of reacting with abifunctional crosslinking agent to form a compound of the invention,such as any one of the to the 12^(th) specific embodiments describedabove; or to form a cell-binding agent conjugate of the invention (suchas those described below). An exemplary linkerless cytotoxic compound ofthe invention includes compound 29b of the 13^(th) specific embodimentabove. The linkerless cytotoxic compounds of the invention arerepresented by any one of the following formulas (I′), (II′), (III′) or(IV′):

or a pharmaceutically acceptable salt thereof, wherein:

-   -   the double line        between N and C represents a single bond or a double bond,        provided that when it is a double bond, X is absent and Y is —H,        or a linear or branched alkyl having 1 to 4 carbon atoms, and        when it is a single bond, X is —H, or an amine protecting        moiety; preferably, the double line        between N and C represents a double bond;    -   Y is —H or a leaving group selected from —OR, —OCOR′, —OCOOR′,        —OCONR′R″, —NR′R″, —NR′COR″, —NR′NR′R″, an optionally        substituted 5- or 6-membered nitrogen-containing heterocycle        (e.g., piperidine, tetrahydropyrrole, pyrazole, morpholine,        etc.), a guanidinum represented by —NR′(C═NH)NR′R″, an amino        acid, or a peptide represented by —NRCOP′, wherein P′ is an        amino acid or a polypeptide containing between 2 to 20 amino        acid units, —SR, —SOR′, —SO₂M, —SO₃M, —OSO₃M, halogen, cyano and        an azido, wherein M is —H or a cation; such as Na⁺ or K⁺.        Preferably, M is —H or Na⁺. Preferably, Y is selected from        —SO₃M, —OH, —OMe, —OEt or —NHOH. More preferably, Y is —SO₃M or        —OH; or,    -   Y is a sulfite (HSO₃, HSO₂ or a salt of HSO₃ ⁻, SO₃ ²⁻ or HSO₂ ⁻        formed with a cation), metabisulfite (H₂S₂O₅ or a salt of S₂O₅        ²⁻ formed with a cation), mono-, di-, tri-, and        tetra-thiophosphate (PO₃SH₃, PO₂S₂H₂, POS₃H₂, PS₄H₂ or a salt of        PO₃S³⁻, PO₂S₂ ³⁻, POS₃ ³⁻ or PS₄ ³⁻ formed with a cation), thio        phosphate ester (R^(i)O)₂PS(OR^(i)), R^(i)S—, R^(i)SO, R^(i)SO₂,        R^(i)SO₃, thiosulfate (HS₂O₃ or a salt of S₂O₃ ²⁻ formed with a        cation), dithionite (HS₂O₄ or a salt of S₂O₄ ²⁻ formed with a        cation), phosphorodithioate (P(═S)(OR^(k′))(S)(OH) or a salt        thereof formed with a cation), hydroxamic acid (R^(k′)C(═O)NOH        or a salt formed with a cation), formaldehyde sulfoxylate        (HOCH₂SO₂ ⁻ or a salt of HOCH₂SO₂ ⁻ formed with a cation, such        as HOCH₂SO₂ ⁻Na⁺) or a mixture thereof, wherein R^(i) is a        linear or branched alkyl having 1 to 10 carbon atoms and is        substituted with at least one substituent selected from        —N(R^(j))₂, —CO₂H, —SO₃H, and —PO₃H; R^(i) can be further        optionally substituted with a substituent for an alkyl described        herein; R^(j) is a linear or branched alkyl having 1 to 6 carbon        atoms; R^(k′) is a linear, branched or cyclic alkyl, alkenyl or        alkynyl having 1 to 10 carbon atoms, aryl, heterocyclyl or        heteroaryl; preferably, Y is an adduct of a bisulfite, a        hydrosulfite, or a metabisulfite, or salts thereof (such as        sodium salt);    -   R, for each occurrence, is independently selected from the group        consisting of —H, an optionally substituted linear, branched or        cyclic alkyl, alkenyl or alkynyl having from 1 to 10 carbon        atoms, a polyethylene glycol unit —(CH₂CH₂O)_(n)—R^(c), an        optionally substituted aryl having 6 to 18 carbon atoms, an        optionally substituted 5- to 18-membered heteroaryl ring        containing one or more heteroatoms independently selected from        nitrogen, oxygen, and sulfur, or an optionally substituted 3- to        18-membered heterocyclic ring containing 1 to 6 heteroatoms        independently selected from O, S, N and P;    -   R′ and R″ are the same or different, and are independently        selected from —H, —OH, —OR, —NHR, —NR₂, —COR, an optionally        substituted linear, branched or cyclic alkyl, alkenyl or alkynyl        having from 1 to 10 carbon atoms, a polyethylene glycol unit        —(CH₂CH₂O)_(n)—R^(c), and an optionally substituted 3- to        18-membered heterocyclic ring having 1 to 6 heteroatoms        independently selected from O, S, N and P;    -   R^(c) is —H or a substituted or unsubstituted linear or branched        alkyl having 1 to 4 carbon atoms;    -   n is an integer from 1 to 24;    -   W is selected from C═O, C═S, CH₂, BH, SO, and SO₂;    -   X′ is selected from the group consisting of —H, —OH, an        amine-protecting group, an optionally substituted linear,        branched or cyclic alkyl, alkenyl or alkynyl having from 1 to 10        carbon atoms, a polyethylene glycol unit —(CH₂CH₂O)_(n)—R^(c),        an optionally substituted aryl having 6 to 18 carbon atoms        (e.g., phenyl), an optionally substituted 5- to 18-membered        heteroaryl ring containing one or more heteroatoms independently        selected from nitrogen, oxygen, and sulfur, and an optionally        substituted 3- to 18-membered heterocyclic ring containing 1 to        6 heteroatoms independently selected from O, S, N and P.        Preferably, X′ is —H, —OH, or -Me. More preferably, X′ is —H;    -   Y′ is selected from the group consisting of —H, an oxo group, an        optionally substituted linear, branched or cyclic alkyl, alkenyl        or alkynyl having from 1 to 10 carbon atoms, an optionally        substituted 6- to 18-membered aryl, an optionally substituted 5-        to 18-membered heteroaryl ring containing one or more        heteroatoms independently selected from nitrogen, oxygen, and        sulfur, an optionally substituted 3- to 18-membered heterocyclic        ring having 1 to 6 heteroatoms. Preferably, Y′ is selected from        —H or oxo. More preferably, Y′ is —H;    -   R₁, R₂, R₃, R₄, R₁′, R₂′, R₃′ and R₄′ are each independently        selected from the group consisting of —H, an optionally        substituted linear, branched or cyclic alkyl, alkenyl or alkynyl        having from 1 to 10 carbon atoms, a polyethylene glycol unit        —(OCH₂CH₂)_(n)—R^(c), halogen, guanidinium [—NH(C═NH)NH₂], —OR,        —NR′R″, —NO₂, —NCO, —NR′COR″, —SR, a sulfoxide represented by        —SOR′, a sulfone represented by —SO₂R′, a sulfonate —SO₃ ⁻M⁺, a        sulfate —OSO₃ ⁻M⁺, a sulfonamide represented by —SO₂NR′R″,        cyano, an azido, —COR′, —OCOR′, and —OCONR′R″. Preferably, 1, 2,        3, or all of R₂, R₃, R₂′ and R₃′ is —H;    -   R₆ is —H, —R, —OR, —SR, —NR′R″, —NO₂, halogen, —OR^(c) or        —SR^(c), wherein R^(c) is —H, a linear or branched alkyl having        1 to 4 carbon atoms. Preferably, R₆ is —OMe or —SMe. Even more        preferably, R₆ is —OMe;

Z and Z′ are independently selected from —(CH₂)_(n′)—,—(CH₂)_(n′)—CR₇R₈—(CH₂)_(na′)—, —(CH₂)_(n′)—NR₉—(CH₂)_(na′)—,—(CH₂)_(n′)—O—(CH₂)_(na′)— and —(CH₂)_(n′)—S—(CH₂)_(na′)—;

-   -   n′ and na′ are same or different, and are selected from 0, 1, 2        and 3;    -   R₇ and R₈ are the same or different, and are each independently        selected from —H, —OH, —SH, —COOH, —NHR′, a polyethylene glycol        unit —(OCH₂CH₂)_(n)—, an amino acid, a peptide unit bearing 2 to        6 amino acids, an optionally substituted linear, branched or        cyclic alkyl having from 1 to 10 carbon atoms;    -   R₉ is independently selected from —H, an optionally substituted        linear, branched or cyclic alkyl having from 1 to 10 carbon        atoms, a polyethylene glycol unit —(OCH₂CH₂)_(n)—;    -   A and A′ are the same or different, and are independently        selected from —O—, oxo (—C(═O)—), —CRR′O—, —CRR′—, —S—, —CRR′S—,        —N(R₅)— and —CRR′N(R₅)—. Preferably, A and A′ are the same or        different, and are selected from —O— and —S—. More preferably, A        and A′ are —O—;    -   R₅ for each occurrence is independently —H or an optionally        substituted linear or branched alkyl having 1 to 10 carbon        atoms;    -   L′, L″, and L′″ are the same or different, and are independently        selected from —H, halogen, an optionally substituted linear,        branched or cyclic alkyl, haloalkyl, alkoxy, haloalkoxy, —NO₂,        or —CN;    -   G is selected from —CH— or —N—.

In certain embodiments, the double line

between N and C represents a single bond, Y is not —H.

In certain embodiments, the double line

between N and C represents a single bond or a double bond, provided thatwhen it is a double bond X is absent and Y is —H, and when it is asingle bond, X is selected from —H, or an amine protecting group(preferably X is —H); W is C═O; R₁, R₂, R₃, R₄, R₁′, R₂′, R₃′, and R₄′are —H; Z and Z′ are —CH₂—; A and A′ are both —O—; W is —(C═O)—; G is—CH—; R₆ is —H, or optionally substituted C1-C10 linear, C1-C10branched, or C3-C7 cyclic alkyl, —O-alkyl, or —O-halo-alkyl, such as—OMe; X′ is selected from the group consisting of —H, —OH, a substitutedor unsubstituted linear, branched or cyclic alkyl, alkenyl or alkynylhaving from 1 to 10 carbon atoms, phenyl, and an amine-protecting group;and Y′ is selected from the group consisting of —H, an oxo group, asubstituted or unsubstituted linear, branched or cyclic alkyl, alkenylor alkynyl having from 1 to 10 carbon atoms.

Preferably, when Y is not —H, Y is selected from —OR, —OCOR′, —SR,—NR′R″, —SO₃M, —SO₂M, or —OSO₃M, wherein M is —H or a cation such asNa⁺. K⁺. Preferably, Y is selected from —H, —OH, —OMe, —OEt, —NHOH or—SO₃M (e.g., Y is —OH, —OMe, —OEt, —NHOH or —SO₃M). Even morepreferably, Y is —H, —OH or —SO₃M (e.g., Y is —OH or —SO₃M), preferablyM is —H or Na⁺.

In certain embodiments, the double line

between N and C represents a single bond or a double bond, provided thatwhen it is a double bond X is absent and Y is —H, and when it is asingle bond, X is selected from —H, or an amine protecting group(preferably X is —H); W is C═O; R₁, R₂, R₃, R₄, R_(1′), R_(2′), R_(3′),R_(4′), X′ and Y′ are —H; Z and Z′ are —CH₂—; A and A′ are both —O—; Wis —(C═O)—; G is —CH—; R₆ is —H, or optionally substituted C1-C10linear, C1-C10 branched, or C3-C7 cyclic alkyl, —O-alkyl, or—O-halo-alkyl, such as —OMe.

The bifunctional crosslinking agents can be any bifunctional linkerknown in the art. For example, the bifunctional linkers can be used formaking the drug-linker compounds are those that form disulfide bonds,thioether bonds, acid labile bonds, photolabile bonds, peptidase labilebonds and esterase labile bonds with the cytotoxic compounds (see forexample, U.S. Pat. Nos. 5,208,020; 5,475,092; 6,441,163; 6,716,821;6,913,748; 7,276,497; 7,276,499; 7,368,565; 7,388,026 and 7,414,073, allof which are incorporated herein by reference). Preferably, thebifunctional crosslinking agents are those that form disulfide bonds,thioether and peptidase labile bonds with the cytotoxic compounds. Otherbifunctional crosslinking agents that can be used in the presentinvention include non-cleavable linkers, such as those described in U.S.publication number US 2005/0169933, or charged linkers or hydrophiliclinkers and are described in US 2009/0274713, US 2010/01293140 and WO2009/134976, each of which is expressly incorporated herein byreference. The bifunctional crosslinking agents that can be used formaking the (drug-linker) compounds of the present invention also includethose described in Thermo Scientific Pierce Crosslinking TechnicalHandbook, the entire teaching of which is incorporated herein byreference.

Synthesis of Cytotoxic Compounds

Representative processes for preparing the cytotoxic dimer compounds ofthe present invention are shown in FIGS. 1-11. The dimers were preparedby reacting a monomer with linker compounds which possess two leavinggroups such as halogen, triflate, mesylate, or tosylate such as thatdescribed for the synthesis of 1c in FIG. 1. Synthesis of representativedimers which bear a thiol or disulfide moiety to enable linkage to cellbinding agents via reducible or non-reducible bonds are shown in FIGS.1-5, 7, 8, and 10. In FIG. 1 a linker containing a short polyethyleneglycol moiety and an alkyl disulfide was prepared through reductiveamination of 1a. Conversion of 1b to its corresponding mesylate andcoupling with the IBD (indolinobenzodiazepine) monomer unit gave dimer1c which was reduced to the mono-imine, converted to the free thiol, andcoupled with 2 to give compound 1g of the present invention. In FIG. 3,a modified form of IBD monomer was prepared and coupled to give a dimerof the present invention in which the reduced imine was converted to alinker. FIG. 4 describes a dimer possessing a short polyethylene glycolmoiety and an amide disulfide which was reduced to thiol 4c andconverted to a reactive ester. FIG. 5 describes the synthesis of pyridyldisulfide containing linker 5e which was converted to the mono-iminethiol 5i of the present invention before being converted to a reactiveester. Synthesis of representative dimers which possess linkers that canreact with cell binding agents are prepared by converting the methylesters to the corresponding reactive esters of a leaving group such as,but not limited to, N-hydroxysuccinimide esters, N-hydroxyphthalimideesters, N-hydroxy sulfo-succinimide esters, para-nitrophenyl esters,pentafluorophenyl esters are shown in FIGS. 6, 9, and 11.

Representative processes for preparing the cytotoxic dimer compounds ofthe present invention suitable for one-step conjugation with a cellbinding agent are shown in FIGS. 1 and 12-19. In all of these examples adimer containing a thiol moiety is reacted with a bifunctionalcrosslinking reagent possessing a reactive group such as, but notlimited to, a thiopyridyl, a maleimide, iodide, bromide, or tosylate onone side and a reactive substituent suitable for reaction with a cellbinding agent such as, but not limited to, N-hydroxysuccinimide esters,N-hydroxyphtalimide esters, N-hydroxy sulfo-succinimide esters,paranitrophenyl esters, pentafluorophenyl esters.

Alternative synthetic processes for preparing representative cytotoxicdimer compounds of the present invention are shown in FIGS. 20-21. InFIG. 20, the synthesis of the mono reduced dimer (i.e., having one iminegroup) is accomplished by a two step coupling method, in which a reducedform of monomer is either initially coupled to the linker followed bycoupling with the IBD monomer or the dimer is prepared using a mixtureof both reduced monomer and the IBD monomer in the coupling with thereactive linker. While the di-reduced dimer is potentially a byproductof the second synthetic pathway previously described, a more directroute is shown in FIG. 21 in which the reduced monomer is coupled toboth with the linker directly.

Cell-Binding Agents

The effectiveness of the conjugates of the invention as therapeuticagents depends on the careful selection of an appropriate cell-bindingagent. Cell-binding agents may be of any kind presently known, or thatbecome known and includes peptides and non-peptides. Generally, thesecan be antibodies (especially monoclonal antibodies), lymphokines,hormones, growth factors, vitamins (such as folate etc., which may bindto a cell surface receptor thereof, e.g., a folate receptor),nutrient-transport molecules (such as transferrin), or any othercell-binding molecule or substance.

In certain embodiments, the cell-binding agents are proteins orpolypeptides, or compounds comprising proteins or polypeptides.Preferably, the proteins or polypeptides comprise one or more Lysresidues with side chain —NH₂ groups. Alternatively or in addition, theproteins or polypeptides comprise one or more Cys residues. The sidechain —SH groups of the Cys residues may be intact, or may be in adisulfide bond that can be reduced. Preferably, reduction of thedisulfide bond(s) does not significantly negatively impact thecell-binding function of the proteins or polypeptides (e.g., in the caseof antibody or antigen-binding portion thereof, reduction of thedisulfide bonds does not substantially increase the dissociation oflight chains/heavy chains).

The Lys side chain —NH₂ groups and/or the Cys side chain —SH groups maybe covalently linked to the linkers, which are in turn linked to thedimer compounds of the invention, thus conjugating the cell-bindingagents to the dimer compounds of the invention. Each protein-basedcell-binding agents may contain multiple Lys side chain —NH₂ groupsand/or the Cys side chain —SH groups available for linking the compoundsof the invention through the bifunctional crosslinkers.

More specific examples of cell-binding agents that can be used include:

polyclonal antibodies;

monoclonal antibodies;

fragments of antibodies such as Fab, Fab′, and F(ab′)₂, Fv, minibodies,diabodies, tribodies, tetrabodies (Parham, J. Immunol. 131:2895-2902(1983); Spring et al. J. Immunol. 113:470-478 (1974); Nisonoff et al.Arch. Biochem. Biophys. 89:230-244 (1960), Kim et al., Mol, CancerTher., 7:2486-2497 (2008), Carter, Nature Revs., 6: 343-357 (2006));

interferons (e.g. α, β, γ);

lymphokines such as IL-2, IL-3, IL-4, IL-6;

hormones such as insulin, TRH (thyrotropin releasing hormone), MSH(melanocyte-stimulating hormone), steroid hormones, such as androgensand estrogens;

growth factors and colony-stimulating factors such as EGF, TGF-alpha,FGF, VEGF, G-CSF, M-CSF and GM-CSF (Burgess, Immunology Today 5:155-158(1984));

transferrin (O'Keefe et al. J. Biol. Chem. 260:932-937 (1985));

vitamins, such as folate;

Protein scaffolds based on a consensus sequence of fibronectin type III(FN3) repeats (also known as Centyrins; See U.S. Patent Publication2010/0255056, incorporated herein by reference);

Designer Ankyrin Repeat Proteins (DARPins; U.S. Patent Application Nos.20040132028; 20090082274; 20110118146; 20110224100, incorporated hereinby reference), C. Zahnd et al. 2010, Cancer Res., 70; 1595-1605,incorporated herein by reference); and,

Fibronectin domain scaffold proteins (Adnectins: US Patent ApplicationNos. 20070082365; 20080139791, incorporated herein by reference).

Monoclonal antibody techniques allow for the production of extremelyspecific cell-binding agents in the form of specific monoclonalantibodies. Particularly well known in the art are techniques forcreating monoclonal antibodies produced by immunizing mice, rats,hamsters or any other mammal with the antigen of interest such as theintact target cell, antigens isolated from the target cell, whole virus,attenuated whole virus, and viral proteins such as viral coat proteins.Sensitized human cells can also be used. Another method of creatingmonoclonal antibodies is the use of phage libraries of scFv (singlechain variable region), specifically human scFv (see e.g., Griffiths etal., U.S. Pat. Nos. 5,885,793 and 5,969,108; McCafferty et al., WO92/01047; Liming et al., WO 99/06587). In addition, resurfacedantibodies disclosed in U.S. Pat. No. 5,639,641 may also be used, as maychimeric antibodies and humanized antibodies. Selection of theappropriate cell-binding agent is a matter of choice that depends uponthe particular cell population that is to be targeted, but in generalhuman monoclonal antibodies are preferred if an appropriate one isavailable.

For example, the monoclonal antibody MY9 is a murine IgG1 antibody thatbinds specifically to the CD33 Antigen {J. D. Griffin et al 8 LeukemiaRes., 521 (1984)} and can be used if the target cells express CD33 as inthe disease of acute myelogenous leukemia (AML). The cell-binding agentmay be any compound that can bind a cell, either in a specific ornon-specific manner. Generally, these can be antibodies (especiallymonoclonal antibodies and antibody fragments), interferons, lymphokines,hormones, growth factors, vitamins, nutrient-transport molecules (suchas transferrin), or any other cell-binding molecule or substance.

Where the cell-binding agent is an antibody, it binds to an antigen thatis a polypeptide and may be a transmembrane molecule (e.g. receptor) ora ligand such as a growth factor. Exemplary antigens include moleculessuch as renin; a growth hormone, including human growth hormone andbovine growth hormone; growth hormone releasing factor; parathyroidhormone; thyroid stimulating hormone; lipoproteins; alpha-1-antitrypsin;insulin A-chain; insulin B-chain; proinsulin; follicle stimulatinghormone; calcitonin; luteinizing hormone; glucagon; clotting factorssuch as factor vmc, factor IX, tissue factor (TF), and von Willebrandsfactor; anti-clotting factors such as Protein C; atrial natriureticfactor; lung surfactant; a plasminogen activator, such as urokinase orhuman urine or tissue-type plasminogen activator (t-PA); bombesin;thrombin; hemopoietic growth factor; tumor necrosis factor-alpha and-beta; enkephalinase; RANTES (regulated on activation normally T-cellexpressed and secreted); human macrophage inflammatory protein(MIP-1-alpha); a serum albumin, such as human serum albumin;Muellerian-inhibiting substance; relaxin A-chain; relaxin B-chain;prorelaxin; mouse gonadotropin-associated peptide; a microbial protein,such as beta-lactamase; DNase; IgE; a cytotoxic T-lymphocyte associatedantigen (CTLA), such as CTLA-4; inhibin; activin; vascular endothelialgrowth factor (VEGF); receptors for hormones or growth factors; proteinA or D; rheumatoid factors; a neurotrophic factor such as bone-derivedneurotrophic factor (BDNF), neurotrophin-3, -4, -5, or -6 (NT-3, NT4,NT-5, or NT-6), or a nerve growth factor such as NGF-β; platelet-derivedgrowth factor (PDGF); fibroblast growth factor such as aFGF and bFGF;fibroblast growth factor receptor 2 (FGFR2), epidermal growth factor(EGF); transforming growth factor (TGF) such as TGF-alpha and TGF-beta,including TGF-β1, TGF-β2, TGF-β3, TGF-β4, or TGF-β5; insulin-like growthfactor-I and -II (IGF-I and IGF-II); des(1-3)-IGF-I (brain IGF-I),insulin-like growth factor binding proteins, melanotransferrin, EpCAM,GD3, FLT3, PSMA, PSCA, MUC1, MUC16, STEAP, CEA, TENB2, EphA receptors,EphB receptors, folate receptor, FOLR1, mesothelin, cripto,alpha_(v)beta₆, integrins, VEGF, VEGFR, EGFR, tarnsferrin receptor,IRTA1, IRTA2, IRTA3, IRTA4, IRTA5; CD proteins such as CD2, CD3, CD4,CD5, CD6, CD8, CD11, CD14, CD19, CD20, CD21, CD22, CD25, CD26, CD28,CD30, CD33, CD36, CD37, CD38, CD40, CD44, CD52, CD55, CD56, CD59, CD70,CD79, CD80. CD81, CD103, CD105, CD134, CD137, CD138, CD152 or anantibody which binds to one or more tumor-associated antigens orcell-surface receptors disclosed in US Publication No. 20080171040 or USPublication No. 20080305044 and are incorporated in their entirety byreference; erythropoietin; osteoinductive factors; immunotoxins; a bonemorphogenetic protein (BMP); an interferon, such as interferon-alpha,-beta, and -gamma; colony stimulating factors (CSFs), e.g., M-CSF,GM-CSF, and G-CSF; interleukins (ILs), e.g., IL-1 to IL-10; superoxidedismutase; T-cell receptors; surface membrane proteins; decayaccelerating factor; viral antigen such as, for example, a portion ofthe HIV envelope; transport proteins; homing receptors; addressins;regulatory proteins; integrins, such as CD11a, CD11b, CD11c, CD18, anICAM, VLA-4 and VCAM; a tumor associated antigen such as HER2, HER3 orHER4 receptor; endoglin, c-Met, c-kit, 1GF1R, PSGR, NGEP, PSMA, PSCA,LGR5, B7H4, and fragments of any of the above-listed polypeptides.

Additionally, GM-CSF, which binds to myeloid cells can be used as acell-binding agent to diseased cells from acute myelogenous leukemia.IL-2 which binds to activated T-cells can be used for prevention oftransplant graft rejection, for therapy and prevention ofgraft-versus-host disease, and for treatment of acute T-cell leukemia.MSH, which binds to melanocytes, can be used for the treatment ofmelanoma, as can antibodies directed towards melanomas. Folic acid canbe used to target the folate receptor expressed on ovarian and othertumors. Epidermal growth factor can be used to target squamous cancerssuch as lung and head and neck. Somatostatin can be used to targetneuroblastomas and other tumor types.

Cancers of the breast and testes can be successfully targeted withestrogen (or estrogen analogues) or androgen (or androgen analogues)respectively as cell-binding agents.

In one embodiment, the cell-binding agent is humanized monoclonalantibodies. In another embodiment, the cell-binding agent is huMy9-6, orother related antibodies, which are described in U.S. Pat. Nos.7,342,110 and 7,557,189 (incorporated herein by reference). In anotherembodiment, the cell-binding agent is an anti-folate receptor antibodydescribed in U.S. Provisional Application Nos. 61/307,797, 61/346,595,61/413,172 and U.S. application Ser. No. 13/033,723 (published as US2012-0009181 A1). The teachings of all these applications areincorporated herein by reference in its entirety.

In certain embodiments, the cell-binding agent may be a monoclonalantibody or antigen-binding portions thereof sharing sequences criticalfor antigen-binding with an antibody disclosed herein, such as huMy9-6or its related antibodies described in U.S. Pat. Nos. 7,342,110 and7,557,189 (incorporated herein by reference). These derivativeantibodies may have substantially the same or identical (1) light chainand/or heavy chain CDR3 regions; (2) light chain and/or heavy chainCDR1, CDR2, and CDR3 regions; or (3) light chain and/or heavy chainregions, compared to an antibody described herein. Sequences withinthese regions may contain conservative amino acid substitutions,including substitutions within the CDR regions. Preferably, there is nomore than 1, 2, 3, 4, or 5 conservative substitutions. In certainembodiments, the derivative antibodies have a light chain region and/ora heavy chain region that is at least about 90%, 95%, 99% or 100%identical to an antibody described herein. These derivative antibodiesmay have substantially the same binding specificity and/or affinity tothe target antigen compared to an antibody described herein. Preferably,the K_(d) and/or k_(off) values of the derivative antibodies are within10-fold (either higher or lower), 5-fold (either higher or lower),3-fold (either higher or lower), or 2-fold (either higher or lower) ofan antibody described herein. These derivative antibodies may be fullyhuman antibodies, or humanized antibodies, or chimeric antibodies. Thederivative antibodies may be produced according to any art-recognizedmethods.

In one embodiment, the anti-folate receptor antibody is a humanizedantibody or antigen binding fragment thereof that specifically binds ahuman folate receptor 1, wherein the antibody comprises: (a) a heavychain CDR1 comprising GYFMN (SEQ ID NO: 1); a heavy chain CDR2comprising RIHPYDGDTFYNQXaa₁FXaa₂Xaa₃ (SEQ ID NO: 2); and a heavy chainCDR3 comprising YDGSRAMDY (SEQ ID NO: 3); and (b) a light chain CDR1comprising KASQSVSFAGTSLMH (SEQ ID NO: 4); a light chain CDR2 comprisingRASNLEA (SEQ ID NO: 5); and a light chain CDR3 comprising QQSREYPYT (SEQID NO: 6); wherein Xaa₁ is selected from K, Q, H, and R; Xaa₂ isselected from Q, H, N, and R; and Xaa₃ is selected from G, E, T, S, A,and V. Preferably, the heavy chain CDR2 sequence comprisesRIHPYDGDTFYNQKFQG (SEQ ID NO: 7).

In another embodiment, the anti-folate receptor antibody is a humanizedantibody or antigen binding fragment thereof that specifically binds thehuman folate receptor 1 comprising the heavy chain having the amino acidsequence of

(SEQ ID NO: 8) QVQLVQSGAEVVKPGASVKISCKASGYTFTGYFMNWVKQSPGQSLEWIGRIHPYDGDTFYNQKFQGKATLTVDKSSNTAHMELLSLTSEDFAVYYCTRYDGSRAMDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

In another embodiment, the anti-folate antibody is a humanized antibodyor antigen binding fragment thereof encoded by the plasmid DNA depositedwith the ATCC on Apr. 7, 2010 and having ATCC deposit nos. PTA-10772 andPTA-10773 or 10774.

In another embodiment, the anti-folate receptor antibody is a humanizedantibody or antigen binding fragment thereof that specifically binds thehuman folate receptor 1 comprising the light chain having the amino acidsequence of

(SEQ ID NO: 9) DIVLTQSPLSLAVSLGQPAIISCKASQSVSFAGTSLMHWYHQKPGQQPRLLIYRASNLEAGVPDRFSGSGSKTDFTLNISPVEAEDAATYYCQQSREYPYTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC; or (SEQ ID NO: 10)DIVLTQSPLSLAVSLGQPAIISCKASQSVSFAGTSLMHWYHQKPGQQPRLLIYRASNLEAGVPDRFSGSGSKTDFTLTISPVEAEDAATYYCQQSREYPYTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC.

In another embodiment the anti-folate receptor antibody is a humanizedantibody or antigen binding fragment thereof that specifically binds thehuman folate receptor 1 comprising the heavy chain having the amino acidsequence of SEQ ID NO: 8, and the light chain having the amino acidsequence of SEQ ID NO: 9 or SEQ ID NO: 10. Preferably, the antibodycomprises the heavy chain having the amino acid sequence of SEQ ID NO: 8and the light chain having the amino acid sequence of SEQ ID NO: 10 (huFOLR1).

In another embodiment, the anti-folate receptor antibody is a humanizedantibody or antigen binding fragment thereof encoded by the plasmid DNAdeposited with the ATCC on Apr. 7, 2010 and having ATCC deposit nos.PTA-10772 and PTA-10773 or 10774.

In another embodiment, the anti-folate receptor antibody is a humanizedantibody or antigen binding fragment thereof comprising a heavy chainvariable domain at least about 90%, 95%, 99% or 100% identical toQVQLVQSGAEVVKPGASVKISCKASGYTFTGYFMNWVKQSPGQSLEWIGRIHPYDGDTFYNQKFQGKATLTVDKSSNTAHMELLSLTSEDFAVYYCTRYDGSRAMDY WGQGTTVTVSS (SEQID NO: 11), and a light chain variable domain at least about 90%, 95%,99% or 100% identical to

(SEQ ID NO: 12) DIVLTQSPLSLAVSLGQPAIISCKASQSVSFAGTSLMHWYHQKPGQQPRLLIYRASNLEAGVPDRFSGSGSKTDFTLNISPVEAEDAATYYCQQSR EYPYTFGGGTKLEIKR; or(SEQ ID NO: 13) DIVLTQSPLSLAVSLGQPAIISCKASQSVSFAGTSLMHWYHQKPGQQPRLLIYRASNLEAGVPDRFSGSGSKTDFTLTISPVEAEDAATYYCQQSR EYPYTFGGGTKLEIKR.

Cell-Binding Agent-Drug Conjugates

The present invention also provides cell-binding agent-drug conjugatescomprising a cell-binding agent linked to one or more cytotoxiccompounds of the present invention via a variety of linkers, including,but not limited to, disulfide linkers, thioether linkers, amide bondedlinkers, peptidase-labile linkers, acid-labile linkers, esterase-labilelinkers.

Representative conjugates of the invention are antibody/cytotoxiccompound, antibody fragment/cytotoxic compound, epidermal growth factor(EGF)/cytotoxic compound, melanocyte stimulating hormone (MSH)/cytotoxiccompound, thyroid stimulating hormone (TSH)/cytotoxic compound,somatostatin/cytotoxic compound, folate/cytotoxic compound,estrogen/cytotoxic compound, estrogen analogue/cytotoxic compound,androgen/cytotoxic compound, and androgen analogue/cytotoxic compound. Arepresentative folate/cytotoxic compound conjugate is depicted below,with the optional —SO₃ ⁻Na⁺ adduct on the imine bond of one of the twodrug monomers. A representative synthesis scheme for this conjugate isshown in FIG. 54.

In a preferred embodiment, the present invention provides conjugatescomprising an indolinobenzodiazepine dimer compound (e.g., compounds offormulas (I)-(IV), (IA)-(IVA) and (IB)-(IVB)) and the cell-binding agentlinked through a covalent bond. The linker can be cleaved at the site ofthe tumor/unwanted proliferating cells to deliver the cytotoxic agent toits target in a number of ways. The linker can be cleaved, for example,by low pH (hydrazone), reductive environment (disulfide), proteolysis(amide/peptide link), or through an enzymatic reaction(esterase/glycosidase).

In a preferred aspect, representative cytotoxic conjugates of theinvention are antibody/indolinobenzodiazepine dimer compound, antibodyfragment/indolinobenzodiazepine dimer compound, epidermal growth factor(EGF)/indolinobenzodiazepine dimer compound, melanocyte stimulatinghormone (MSH)/indolinobenzodiazepine dimer compound, thyroid stimulatinghormone (TSH)/indolinobenzodiazepine dimer compound,somatostatin/indolinobenzodiazepine dimer compound,folate/indolinobenzodiazepine dimer compound,estrogen/indolinobenzodiazepine dimer compound, estrogenanalogue/indolinobenzodiazepine dimer compound, prostate specificmembrane antigen (PSMA) inhibitor/indolinobenzodiazepine dimer compound,matriptase inhibitor/indolinobenzodiazepine dimer compound, designedankyrin repeat proteins (DARPins)/indolinobenzodiazepine dimer compound,androgen/indolinobenzodiazepine dimer compound, and androgenanalogue/indolinobenzodiazepine dimer compound.

Thus in the fourteenth specific embodiment, the invention provides aconjugate comprising: a cytotoxic compound and a cell binding agent(CBA), wherein the cytotoxic compound comprises a linking group whichcovalently links the cytotoxic compound to the CBA, and wherein thecytotoxic compound is represented by any one of the following formulas:

or a pharmaceutically acceptable salt thereof, wherein:

-   -   the double line        between N and C represents a single bond or a double bond,        provided that when it is a double bond X is absent and Y is —H,        or a linear or branched alkyl having 1 to 4 carbon atoms, and        when it is a single bond, X is —H, the linking group, or an        amine protecting moiety;    -   Y is —H or a leaving group selected from —OR, —OCOR′, —OCOOR′,        —OCONR′R″, —NR′R″, —NR′COR″, —NR′NR′R″, an optionally        substituted 5 or 6-membered nitrogen-containing heterocycle        (e.g., piperidine, tetrahydropyrrole, pyrazole, morpholine), a        guanidinum represented by —NR′(C═NH)NR′R″, an amino acid, or a        peptide represented by —NRCOP′, wherein P′ is an amino acid or a        polypeptide containing between 2 to 20 amino acid units, —SR,        —SOR′, —SO₂M, —SO₃M, —OSO₃M, halogen, cyano and an azido; or,    -   Y is a sulfite (HSO₃, HSO₂ or a salt of HSO₃ ⁻, SO₃ ²⁻or HSO₂ ⁻        formed with a cation), metabisulfite (H₂S₂O₅ or a salt of S₂O₅        ²⁻ formed with a cation), mono-, di-, tri-, and        tetra-thiophosphate (PO₃SH₃, PO₂S₂H₂, POS₃H₂, PS₄H₂ or a salt of        PO₃S³⁻, PO₂S₂ ³⁻, POS₃ ³⁻ or PS₄ ³⁻ formed with a cation), thio        phosphate ester (R^(i)O)₂PS(OR^(i)), R^(i)S—, R^(i)SO, R^(i)SO₂,        R^(i)SO₃, thiosulfate (HS₂O₃ or a salt of S₂O₃ ²⁻formed with a        cation), dithionite (HS₂O₄ or a salt of S₂O₄ ²⁻ formed with a        cation), phosphorodithioate (P(═S)(OR^(k′))(S)(OH) or a salt        thereof formed with a cation), hydroxamic acid (R^(k′)C(═O)NOH        or a salt formed with a cation), formaldehyde sulfoxylate        (HOCH₂SO₂ ⁻ or a salt of HOCH₂SO₂ ⁻ formed with a cation, such        as HOCH₂SO₂ ⁻Na⁺) or a mixture thereof, wherein R^(i) is a        linear or branched alkyl having 1 to 10 carbon atoms and is        substituted with at least one substituent selected from        —N(R^(j))₂, —CO₂H, —SO₃H, and —PO₃H; R^(i) can be further        optionally substituted with a substituent for an alkyl described        herein; R^(j) is a linear or branched alkyl having 1 to 6 carbon        atoms; R^(k′) is a linear, branched or cyclic alkyl, alkenyl or        alkynyl having 1 to 10 carbon atoms, aryl, heterocyclyl or        heteroaryl; preferably, Y is an adduct of a bisulfite, a        hydrosulfite, or a metabisulfite, or salts thereof (such as        sodium salt);    -   M is —H or a cation;    -   R, for each occurrence, is independently selected from the group        consisting of —H, an optionally substituted linear, branched or        cyclic alkyl, alkenyl or alkynyl having from 1 to 10 carbon        atoms, a polyethylene glycol unit —(CH₂CH₂O)_(n)—R^(c), an        optionally substituted aryl having 6 to 18 carbon atoms, an        optionally substituted 5- to 18-membered heteroaryl ring        containing one or more heteroatoms independently selected from        nitrogen, oxygen, and sulfur, or an optionally substituted 3- to        18-membered heterocyclic ring containing 1 to 6 heteroatoms        independently selected from O, S, N and P;    -   R′ and R″ are each independently selected from —H, —OH, —OR,        —NHR, —NR₂, —COR, an optionally substituted linear, branched or        cyclic alkyl, alkenyl or alkynyl having from 1 to 10 carbon        atoms, a polyethylene glycol unit —(CH₂CH₂O)_(n)—R^(c), and an        optionally substituted 3- to 18-membered heterocyclic ring        having 1 to 6 heteroatoms independently selected from O, S, N        and P;    -   R^(c) is —H or a substituted or unsubstituted linear or branched        alkyl having 1 to 4 carbon atoms, or the linking group;    -   n is an integer from 1 to 24;    -   W is selected from C═O, C═S, CH₂, BH, SO and SO₂;    -   X′ is selected from —H, an amine-protecting group, the linking        group, an optionally substituted linear, branched or cyclic        alkyl, alkenyl or alkynyl having from 1 to 10 carbon atoms, a        polyethylene glycol unit —(CH₂CH₂O)_(n)—R^(c), an optionally        substituted aryl having 6 to 18 carbon atoms, an optionally        substituted 5- to 18-membered heteroaryl ring containing one or        more heteroatoms independently selected from nitrogen, oxygen,        and sulfur, and an optionally substituted 3- to 18-membered        heterocyclic ring containing 1 to 6 heteroatoms independently        selected from O, S, N and P;    -   Y′ is selected from —H, an oxo group, the linking group, an        optionally substituted linear, branched or cyclic alkyl, alkenyl        or alkynyl having from 1 to 10 carbon atoms, an optionally        substituted 6- to 18-membered aryl, an optionally substituted 5-        to 18-membered heteroaryl ring containing one or more        heteroatoms independently selected from nitrogen, oxygen, and        sulfur, an optionally substituted 3- to 18-membered heterocyclic        ring having 1 to 6 heteroatoms;    -   R₁, R₂, R₃, R₄, R₁′, R₂′, R₃′ and R₄′ are each independently        selected from the group consisting of —H, an optionally        substituted linear, branched or cyclic alkyl, alkenyl or alkynyl        having from 1 to 10 carbon atoms, a polyethylene glycol unit        —(OCH₂CH₂)_(n)—R^(c), halogen, guanidinium [—NH(C═NH)NH₂], —OR,        —NR′R″, —NO₂, —NCO, —NR′COR″, —SR, a sulfoxide represented by        —SOR′, a sulfone represented by —SO₂R′, a sulfonate —SO₃ ⁻M⁺, a        sulfate —OSO₃ ⁻M⁺, a sulfonamide represented by —SO₂NR′R″,        cyano, an azido, —COR′, —OCOR′, —OCONR′R″ and the linking group;    -   R₆ is —H, —R, —OR, —SR, —NR′R″, —NO₂, halogen or the linking        group; Z and Z′ are independently selected from —(CH₂)_(n′)—,        —(CH₂)_(n′)—CR₇R₈—(CH₂)_(na′)—, —(CH₂)_(n′)—NR₉—(CH₂)_(na′)—,        —(CH₂)_(n)′—O—(CH₂)_(na′)— and —(CH₂)_(n′)—S—(CH₂)_(na′)—;    -   n′ and na′ are the same or different, and are selected from 0,        1, 2 and 3;    -   R₇ and R₈ are the same or different, and are each independently        selected from —H, —OH, —SH, —COOH, —NHR′, a polyethylene glycol        unit —(OCH₂CH₂)_(n)—, an amino acid, a peptide unit bearing 2 to        6 amino acids, an optionally substituted linear, branched or        cyclic alkyl having from 1 to 10 carbon atoms;    -   R₉ is independently selected from —H, an optionally substituted        linear, branched or cyclic alkyl having from 1 to 10 carbon        atoms, a polyethylene glycol unit —(OCH₂CH₂)_(n)—;    -   A and A′ are the same or different, and are independently        selected from —O—, oxo (—C(═O)—), —CRR′O—, —CRR′—, —S—, —CRR′S—,        —NR₅ and —CRR′N(R₅)—;    -   R₅ for each occurrence is independently —H or an optionally        substituted linear or branched alkyl having 1 to 10 carbon        atoms;    -   D and D′ are the same or different, and are independently absent        or selected from the group consisting of an optionally        substituted linear, branched or cyclic alkyl, alkenyl or alkynyl        having 1 to 10 carbon atoms, an amino acid, a peptide bearing 2        to 6 amino acids, and a polyethylene glycol unit        (—OCH₂CH₂)_(n)—;    -   L is absent, the linking group, a polyethylene glycol unit        (—OCH₂CH₂)_(n)—, a linear, branched or cyclic alkyl or alkenyl        having 1 to 10 carbon atoms, a phenyl group, a 3- to 18-membered        heterocyclic ring or a 5- to 18-membered heteroaryl ring having        1 to 6 heteroatoms independently selected from O, S, N and P,        wherein the alkyl or alkenyl is optionally substituted with the        linking group; phenyl or heterocyclic or heteroaryl ring can be        optionally substituted, wherein the substituent can be the        linking group.

In certain embodiments, X is not the linking group. In certainembodiments, the double line

between N and C represents a single bond, Y is not —H.

In certain embodiments, Y is a leaving group selected from —OR, —OCOR′,—OCOOR′, —OCONR′R″, —NR′R″, —NR′COR″, —NR′NR′R″, an optionallysubstituted 5 or 6-membered nitrogen-containing heterocycle (e.g.,piperidine, tetrahydropyrrole, pyrazole, morpholine, etc.), a guanidinumrepresented by —NR′(C═NH)NR′R″, an amino acid, or a peptide representedby —NRCOP′, wherein P′ is an amino acid or a polypeptide containingbetween 2 to 20 amino acid units, —SR, —SOR′, —SO₂M, —SO₃M, —OSO₃M,halogen, cyano and an azido.

In certain embodiments, the compound is not any one of the followingcompounds:

In certain embodiments, the conjugates of the invention include thefollowing:

wherein:

-   -   CBA is the cell-binding agent, r is an integer from 1 to 10, Y        is —H, an adduct of a bisulfite, a hydrosulfite, or a        metabisulfite, or salts thereof, or —SO₃M, and M is —H or a        pharmaceutically acceptable cation.

In certain embodiments, L is absent, or is selected from an optionallysubstituted phenyl group and an optionally substituted pyridyl group,wherein the phenyl and the pyridyl group bears the linking group, or Lis an amine group bearing the linking group (i.e., —N(linking group)-),or L is a linear, branched or cyclic alkyl or alkenyl having from 1 to 6carbon atoms and bearing the linking group.

In the fifteenth specific embodiment, the compound is represented by anyone of the following formulas:

wherein:

-   -   L′, L″, and L′″ are the same or different, and are independently        selected from —H, an optionally substituted linear, branched or        cyclic alkyl, alkenyl or alkynyl having from 1 to 10 carbon        atoms, a polyethylene glycol unit —(OCH₂CH₂)_(n)—R^(c), halogen,        guanidinium [—NH(C═NH)NH₂], —OR, —NR′R″, —NO₂, —NR′COR″, —SR, a        sulfoxide represented by —SOR′, a sulfone represented by —SO₂R′,        a sulfonate —SO₃M, a sulfate —OSO₃M, a sulfonamide represented        by —SO₂NR′R″, cyano, an azido, —COR′, —OCOR′, —OCONR′R″ and the        linking group, provided only one of L′, L″, and L″ is the        linking group; and    -   G is selected from —CH— or —N—. The remaining groups are as        described in the fourteenth specific embodiment above.

In certain embodiments, one of L′, L″, or L′″ is the linking group,while the others are —H. Preferably, L′ is the linking group, and L″ andL′″ are —H.

In certain embodiments, A and A′ are both —O—, R₆ is —OMe, and G is—CH—.

In a sixteenth specific embodiment, L′ is represented by the followingformula:

—W′—R^(x)—V—R^(y)-J,

wherein:

-   -   W′ and V are the same or different, and are each independently        absent, or selected from —CR^(e)R^(e)—, —O—, —O—C(═O)—,        —C(═O)—O—, —S—, —SO—, —SO₂—, —CH₂—S—, —CH₂O—, —CH₂NR^(e)—,        —O—(C═O)O—, —O—(C═O)N(R^(e))—, —N(R^(e))—, —N(R^(e))—C(═O)—,        —C(═O)—N(R^(e))—, —N(R^(e))—C(═O)O—, —N(C(═O)R^(e))C(═O)—,        —N(C(═O)R^(e)—, —(O—CH₂—CH₂)_(n)—, —SS—, or —C(═O)—, or an amino        acid, or a peptide having 2 to 8 amino acids;    -   R^(x) and R^(y) are the same or different, and are each        independently absent or an optionally substituted linear,        branched or cyclic alkyl, alkenyl, or alkynyl having 1 to 10        carbon atoms, an aryl bearing 6 to 10 carbon atoms or a 3- to        8-membered hetereocyclic ring bearing 1 to 3 heteroatoms        selected from O, N or S;

R^(e) and R^(e′) are the same or different, and are selected from —H, alinear, branched or cyclic alkyl, alkenyl, or alkynyl having 1 to 10carbon atoms or —(CH₂—CH₂—O)_(n)—R^(k), wherein R^(k) is a —H, a linear,branched cyclic alkyl having 1 to 6 carbon atoms, optionally bearing asecondary amino (e.g., —NHR¹⁰¹) or tertiary amino (—NR¹⁰¹R¹⁰²) group ora 5- or 6-membered nitrogen containing heterocycle, such as piperidineor morpholine, wherein R¹⁰¹ and R¹⁰² are each independently a linear,branched, or cyclic alkyl, alkenyl or alkynyl having 1 to 10 carbonatoms; preferably, R¹⁰¹ and R¹⁰² are each independently a linear orbranched alkyl having 1 to 6 carbon atoms;

-   -   n is an integer from 1 to 24; and    -   J is covalently linked to the CBA, and is selected from a        succinimide, a acetamido, —S—, —SS—, —CH₂S—, —CH(Me)S—,        —C(Me)₂S—, —NR^(c1)—, —CH₂NR^(c1)—, —NR^(c1)N—, and —C(═O)—,        wherein R^(c1) is —H or a substituted or unsubstituted linear or        branched alkyl having 1 to 4 carbon atoms.

In certain embodiments, J is —S—, —SS—, a succinimide, or —C(═O)—.

In certain embodiments, R^(e′) is —H or -Me; R^(e) is a linear orbranched alkyl having 1 to 6 carbon atoms or —(CH₂—CH₂—O)_(n)—R^(k); nis an integer from 2 to 8; and R^(k) is —H, -Me or —CH₂CH₂—NMe₂, and theremainder of the variables are as described above in the fifteenthspecific embodiment.

In certain embodiments, V is an amino acid or a peptide having 2 to 8amino acids.

In certain embodiments, V is valine-citrulline, gly-gly-gly, orala-leu-ala-leu.

In certain embodiments,

-   -   W′ is —O—, —N(R^(e))— or —N(R^(e))—C(═O)—;    -   R^(e) is H, a linear or branched alkyl having 1 to 4 carbon        atoms, or —(CH₂—CH₂—O)_(n)—R^(k);    -   R^(x) is a linear or branched alkyl having 1 to 6 carbon atoms;    -   V is absent, —(O—CH₂—CH₂)_(n)—, —C(═O)—NH—, —S—, —NH—C(═O)—;    -   R^(y) is absent or a linear or branched alkyl having 1 to 4        carbon atoms; and    -   J is —S—, —SS—, or —C(═O)—, and the remaining groups are as        defined in the sixteenth specific embodiment.

In certain embodiments,

-   -   W′ is —O—, —N(R^(e))— or —N(R^(e))—C(═O)—;    -   R^(e) is —H, -Me, or —(CH₂—CH₂—O)_(n)-Me;    -   n is an integer from 2 to 6;    -   R^(x) is linear or branched alkyl bearing 1 to 6 carbon atoms;    -   V and R^(y) are absent; and    -   J is —C(═O)—. The remaining groups are as defined in the        sixteenth specific embodiment.

In a seventeenth specific embodiment, L′ in the sixteenth specificembodiment is represented by the following formula:

—W′—[CR_(1″)R_(2″)]_(a)—V—[Cy]₀₋₁—[CR_(3″)R_(4″)]_(b)—C(═O)—,

wherein:

-   -   R_(1″), R_(2″), and R_(3″) are each independently —H or a linear        or branched alkyl bearing 1 to 4 carbon atoms, preferably -Me;    -   R_(4″) is —H, a linear or branched alkyl bearing 1 to 4 carbon        atoms (preferably -Me), —SO₃H, or —SO₃ ⁻M⁺, wherein M⁺ is a        pharmaceutically acceptable cation;    -   a is an integers from 0-5 (e.g., from 0 to 2, 3, 4, or 5), and b        is an integer from 0-6 (e.g., from 0 to 3, 4, 5, or 6); and,    -   Cy is an optionally substituted 5-membered heterocyclic ring        bearing an N heteroatom, preferably Cy is

In certain embodiments, such as in the sixteenth or the seventeenthspecific embodiment, W′ is —N(R^(e))—.

In certain embodiments, such as in the sixteenth or the seventeenthspecific embodiment, R^(e) is —(CH₂—CH₂—O)₂₋₆—R^(k), wherein R^(k) is alinear or branched alkyl having 1 to 6 carbon atoms.

In certain embodiments, such as in the sixteenth or the seventeenthspecific embodiment, V is —S— or —SS—.

In an eighteenth specific embodiment, L′ in the sixteenth or theseventeenth specific embodiment is represented by the following formula:

NR^(e)—[CR_(1″)R_(2″)]_(a)—S—[CR_(3″)R_(4″)]_(b)—C(═O)—.

In certain embodiments, such as in the sixteenth to eighteenth specificembodiments, the conjugate is:

-   -   wherein r is an integer from 1 to 10, Y is —H or —SO₃M (e.g., Y        is —SO₃M), and M is —H or a pharmaceutically acceptable cation.

In certain embodiments, such as in the sixteenth to eighteenth specificembodiments, the antibody is huMy9-6.

In a nineteenth specific embodiment, L′ in the sixteenth or theseventeenth specific embodiment is represented by the following formula:

NR^(e)—[CR_(1″)R_(2″)]_(a)—S-Cy-[CR_(3″)R_(4″)]_(b)—C(═O)—.

In certain embodiments, such as in the sixteenth, seventeenth, and thenineteenth specific embodiments, the conjugate is:

-   -   wherein r is an integer from 1 to 10, Y is —H or —SO₃M (e.g., Y        is —SO₃M), and M is —H or a pharmaceutically acceptable cation.

In certain embodiments, such as in the sixteenth, seventeenth, and thenineteenth specific embodiments, the antibody is huMy9-6.

In a twentieth specific embodiment, the compound is represented by thefollowing formula:

wherein:

-   -   W′ is absent, or selected from —O—, —N(R^(e))—,        —N(R^(e))—C(═O)—, —N(C(═O)R^(e))—, —S—, —CH₂—S—, or —CH₂NR^(e)—;    -   R^(x) is absent or selected from a linear, branched or cyclic        alkyl having 1 to 10 carbon atoms;    -   R^(e) is —H, a linear, branched or cyclic alkyl, alkenyl or        alkynyl having 1 to 10 carbon atoms or —(CH₂—CH₂—O)_(n)—R^(k),        wherein R^(k) is a —H, a linear, branched cyclic alkyl having 1        to 6 carbon atoms, optionally bearing a secondary amino (e.g.,        —NHR¹⁰¹) or tertiary amino (—NR¹⁰¹R¹⁰²) group or a 5 or        6-membered nitrogen containing heterocycle, such as piperidine        or morpholine, wherein 8101 and R¹⁰² are each independently a        linear, branched, or cyclic alkyl, alkenyl or alkynyl having 1        to 10 carbon atoms;    -   Z^(s) is linked to the CBA, and is either a bond, or —SR^(m)—;    -   R^(m) is R^(d) or a substituted linear or branched alkyl having        1 to 4 carbon atoms bearing a reactive ester, selected from        N-hydroxysuccinimide esters, N-hydroxyphtalimide esters,        N-hydroxy sulfo-succinimide esters, para-nitrophenyl esters,        dinitrophenyl esters, and pentafluorophenyl esters;    -   R^(d) is selected from phenyl, nitrophenyl, dinitrophenyl,        carboxynitrophenyl, pyridyl or nitropyridyl; and    -   n is an integer from 1 to 24; and the remainder of the variables        are as described above in the eighth or the fifteenth specific        embodiment.

In a twenty-first specific embodiment, the compound is represented bythe following formula:

wherein:

-   -   W′ is absent, or selected from —O—, —N(R^(e))—,        —N(R^(e))—C(═O)—, —N(C(═O)R^(e))—, —S—, —CH₂—S—, or —CH₂NR^(e)—;    -   R^(x) is absent or selected from a linear, branched or cyclic        alkyl having 1 to 10 carbon atoms;    -   R^(e) is —H, a linear, branched or cyclic alkyl, alkenyl or        alkynyl having 1 to 10 carbon atoms or —(CH₂—CH₂—O)_(n)—R^(k),        wherein R^(k) is a —H, a linear, branched cyclic alkyl having 1        to 6 carbon atoms, optionally bearing a secondary amino (e.g.,        —NHR¹⁰¹) or tertiary amino (—NR¹⁰¹R¹⁰²) group or a 5 or        6-membered nitrogen containing heterocycle, such as piperidine        or morpholine, wherein 8101 and R¹⁰² are each independently a        linear, branched, or cyclic alkyl, alkenyl or alkynyl having 1        to 10 carbon atoms;    -   n is an integer from 2 to 6;    -   Z^(s) is linked to the CBA, and is selected from:        -   a bond;

wherein:

-   -   q is an integer from 1 to 5; and,    -   M is —H or a cation, such as Na⁺ or K⁺.

In certain embodiments, Z^(s) is represented by any one of the followingformulas:

In certain embodiments, W′ is —N(R^(e))—.

In certain embodiments, R^(e) is —(CH₂—CH₂—O)_(n)—R^(k), wherein R^(k)is a —H, a linear, branched cyclic alkyl having 1 to 6 carbon atoms.

In certain embodiments, R^(k) is —H or -Me, n is 4, and q is 2.

In certain embodiments, R^(x) is a linear or branched alkyl having 1 to6 carbon atoms.

In certain embodiments, R^(x) is —(CH₂)_(p)—(CR^(f)R^(g))—, whereinR^(f) and R^(g) are each independently selected from H or a linear orbranched alkyl having 1 to 4 carbon atoms; and p is 0, 1, 2 or 3.

In certain embodiments, R^(f) and R^(g) are the same or different, andare selected from —H and -Me; and p is 1.

In a twenty-second specific embodiment, the conjugate of formula (VIII),(IX), (X) and (XI) described in the twenty-first specific embodiment,the variables are as described below:

-   -   the double line        between N and C represents a single bond or a double bond,        provided that when it is a double bond X is absent and Y is —H,        and when it is a single bond, X is —H; Y is —H, —OH or —SO₃M        (e.g., Y is —OH or —SO₃M);    -   M is —H or a pharmaceutically acceptable cation (e.g., Na⁺);    -   X′ and Y′ are both —H;    -   A and A′ are both —O—;    -   R₆ is —OMe; and    -   R^(x) is a linear or branched alkyl having 1 to 6 carbon atoms.

In a twenty-third specific embodiment, for compounds of formula (IB),(IIB), (IIIB) and (IVB) described in the twentieth specific embodiment,the variables are as described below:

-   -   the double line        between N and C represents a single bond or a double bond,        provided that when it is a double bond X is absent and Y is —H,        and when it is a single bond, X is —H; Y is —H, —OH or —SO₃M        (e.g., Y is —OH or —SO₃M);    -   M is —H or Na⁺;    -   X′ and Y′ are both —H;    -   A and A′ are both —O—;    -   R₆ is —OMe;    -   R^(x) is a linear or branched alkyl having 1 to 6 carbon atoms.

Preferably, R^(x) is —(CH₂)_(p)—(CR^(f)R^(g))—, wherein R^(f) and R^(g)are each independently selected from —H or a linear or branched alkylhaving 1 to 4 carbon atoms; p is 0, 1, 2 or 3. More preferably, R^(f)and R^(g) are the same or different, and are selected from —H and -Me;and p is 1.

In any of the specific embodiments for the conjugate of the inventionabove, such as the fourteenth to the twenty-third specific embodiments,the double line

between N and C may represent a double bond.

In any of the specific embodiments for the conjugate of the inventionabove, such as the fourteenth to the twenty-third specific embodiments,the double line

between N and C may represent a single bond, X is —H, the linking group,or an amine protecting group (e.g., X is —H); and Y is —H or selectedfrom —OR, —OCOR′, —SR, —NR′R″, an optionally substituted 5- or6-membered nitrogen-containing heterocycle, —SO₃M, —SO₂M and a sulfate—OSO₃M. In certain embodiments, Y is not —H.

In certain embodiments, Y is selected from —H, —SO₃M, —OH, —OMe, —OEt or—NHOH (e.g., Y is —SO₃M, —OH, —OMe, —OEt or —NHOH).

In certain embodiments, Y is —H, —SO₃M or —OH (e.g., Y is —SO₃M or —OH).

In certain embodiments, M is —H, Na⁺ or K⁺.

In any of the specific embodiments for the conjugate of the inventionabove, such as the fourteenth to the twenty-third specific embodiments,W, when present, is C═O.

In any of the specific embodiments for the conjugate of the inventionabove, such as the fourteenth to the twenty-third specific embodiments,Z and Z′, when present, are —CH₂—.

In any of the specific embodiments for the conjugate of the inventionabove, such as the fourteenth to the twenty-third specific embodiments,X′ is selected from the group consisting of —H, —OH, an optionallysubstituted linear, branched or cyclic alkyl, alkenyl or alkynyl havingfrom 1 to 10 carbon atoms, phenyl, the linking group, and anamine-protecting group.

In certain embodiments, X′ is —H, —OH, -Me or the linking group.

In certain embodiments, X′ is —H.

In any of the specific embodiments for the conjugate of the inventionabove, such as the fourteenth to the twenty-third specific embodiments,Y′ is selected from the group consisting of —H, an oxo group, asubstituted or unsubstituted linear, branched or cyclic alkyl, alkenylor alkynyl having from 1 to 10 carbon atoms.

In certain embodiments, Y′ is —H or oxo.

In certain embodiments, Y′ is —H.

In any of the specific embodiments for the conjugate of the inventionabove, such as the fourteenth to the twenty-third specific embodiments,A and A′ are the same or different, and are selected from —O—, —S—,—N(R₅)—, and oxo (C═O).

In certain embodiments, A and A′ are the same or different, and areselected from —O— and —S—.

In certain embodiments, A and A′ are —O—.

In any of the specific embodiments for the conjugate of the inventionabove, such as the fourteenth to the twenty-third specific embodiments,D and D′, when present, are the same or different, and are independentlyselected from a polyethylene glycol unit (—OCH₂CH₂)_(n), wherein n is aninteger from 1 to 24, an amino acid, a peptide bearing 2 to 6 aminoacids, or a linear, branched or cyclic alkyl, alkenyl or alkynyl having1 to 10 carbon atoms, wherein the alkyl, alkenyl and alkynyl areoptionally substituted with one or more substituents independentlyselected from the group consisting of halogen, —OR, —NR′COR″, —SR and—COR′.

In certain embodiments, D and D′ are linear or branched alkyl bearing 1to 4 carbon atoms.

In a twenty-fourth specific embodiment, the conjugate of the presentinvention as described in the fourteenth, fifteenth, or the twenty-firstspecific embodiment is represented by the following:

-   -   the double line        between N and C represents a double bond;    -   Y is —H;    -   W is C═O;    -   R₁, R₂, R₁′, R₂′, R₄ and R₄′ are —H; one of R₃, or R₃′ is        optionally the linking group and the other is —H;    -   R₆ is —OMe;    -   Z and Z′ are —CH₂;    -   X′ is —H;    -   Y′ is —H; and    -   A and A′ are —O—.

In certain embodiments, the conjugate of any one of the describedembodiments, such as the fourteenth to the twenty-fourth specificembodiments, may comprise 1-10 cytotoxic compounds, 2-9 cytotoxiccompounds, 3-8 cytotoxic compounds, 4-7 cytotoxic compounds, or 5-6cytotoxic compounds, each cytotoxic compound comprising the linkinggroup linking the cytotoxic compound to the CBA, and each cytotoxiccompound on the conjugate is the same.

In any of the conjugates embodiments, such as the fourteenth to thetwenty-fourth specific embodiments, the cell-binding agent may bind totarget cells selected from tumor cells, virus infected cells,microorganism infected cells, parasite infected cells, autoimmune cells,activated cells, myeloid cells, activated T-cells, B cells, ormelanocytes; cells expressing the CD4, CD6, CD19, CD20, CD22, CD30,CD33, CD37, CD38, CD40, CD44, CD56, EpCAM, CanAg, CALLA, or Her-2antigens; Her-3 antigens; or cells expressing insulin growth factorreceptor, epidermal growth factor receptor, and folate receptor.

In any of the conjugates embodiments, such as the fourteenth to thetwenty-fourth specific embodiments, the cell-binding agent may be anantibody, a single chain antibody, an antibody fragment thatspecifically binds to the target cell, a monoclonal antibody, a singlechain monoclonal antibody, or a monoclonal antibody fragment thatspecifically binds to a target cell, a chimeric antibody, a chimericantibody fragment that specifically binds to the target cell, a domainantibody, a domain antibody fragment that specifically binds to thetarget cell, a lymphokine, a hormone, a vitamin, a growth factor, acolony stimulating factor, or a nutrient-transport molecule.

The antibody may be a resurfaced antibody, a resurfaced single chainantibody, or a resurfaced antibody fragment.

The antibody may be a monoclonal antibody, a single chain monoclonalantibody, or a monoclonal antibody fragment thereof.

The antibody may be a humanized antibody, a humanized single chainantibody, or a humanized antibody fragment.

The invention further provides a pharmaceutical composition comprisingany of the conjugates described herein, and a pharmaceuticallyacceptable carrier.

The invention further provides a drug-linker compound comprising any ofthe subject compound covalently linked to a bifunctional linker.

The invention additional provides a conjugate comprising any of thesubject compounds, or the subject drug-linker compounds, linked to acell-binding agent.

The invention further provides a method of inhibiting abnormal cellgrowth or treating a proliferative disorder, an autoimmune disorder,destructive bone disorder, infectious disease, viral disease, fibroticdisease, neurodegenerative disorder, pancreatitis or kidney disease in amammal comprising administering to the mammal a therapeuticallyeffective amount of any of the compounds (with or without any linkergroup) or conjugates of the invention, and, optionally, a secondchemotherapeutic agent.

In certain embodiments, the compound or the conjugate is:

-   -   wherein r is an integer from 1 to 10, Y is —H or —SO₃M (e.g., Y        is —SO₃M), and M is —H or a pharmaceutically acceptable cation.

In certain embodiments, the second chemotherapeutic agent isadministered to the mammal sequentially or consecutively.

In certain embodiments, the method is for treating a condition selectedfrom cancer, rheumatoid arthritis, multiple sclerosis, graft versus hostdisease (GVHD), transplant rejection, lupus, myositis, infection, andimmune deficiency.

In certain embodiments, the method or conjugate is for treating acancer.

In certain embodiments, the cancer is selected from breast cancer, coloncancer, brain cancer, prostate cancer, kidney cancer, pancreatic cancer,ovarian cancer, head and neck cancer, melanoma, colorectal cancer,gastric cancer, squamous cancer, small-cell lung cancer, non small-celllung cancer, testicular cancer, Merkel cell carcinoma, glioblastoma,neuroblastoma, cancers of lymphatic organs and hematological malignancyincluding Leukemia (Acute lymphoblastic leukemia (ALL), Acutemyelogenous leukemia (AML), Chronic lymphocytic leukemia (CLL), Chronicmyelogenous leukemia (CML), Acute monocytic leukemia (AMOL), Hairy cellleukemia (HCL), T-cell prolymphocytic leukemia (T-PLL), Large granularlymphocytic leukemia, Adult T-cell leukemia), Lymphoma (smalllymphocytic lymphoma (SLL), Hodgkin's lymphomas (Nodular sclerosis,Mixed cellularity, Lymphocyte-rich, Lymphocyte depleted or not depleted,and Nodular lymphocyte-predominant Hodgkin lymphoma), Non-Hodgkin'slymphomas (all subtypes), Chronic lymphocytic leukemia/Small lymphocyticlymphoma, B-cell prolymphocytic leukemia, Lymphoplasmacytic lymphoma(such as Waldenström macroglobulinemia), Splenic marginal zone lymphoma,Plasma cell neoplasms (Plasma cell myeloma, Plasmacytoma, Monoclonalimmunoglobulin deposition diseases, Heavy chain diseases), Extranodalmarginal zone B cell lymphoma (MALT lymphoma), Nodal marginal zone Bcell lymphoma (NMZL), Follicular lymphoma, Mantle cell lymphoma, Diffuselarge B cell lymphoma, Mediastinal (thymic) large B cell lymphoma,Intravascular large B cell lymphoma, Primary effusion lymphoma, Burkittlymphoma/leukemia, T cell prolymphocytic leukemia, T cell large granularlymphocytic leukemia, Aggressive NK cell leukemia, Adult T cellleukemia/lymphoma, Extranodal NK/T cell lymphoma (nasal type),Enteropathy-type T cell lymphoma, Hepatosplenic T cell lymphoma, BlasticNK cell lymphoma, Mycosis fungoides/Sezary syndrome, Primary cutaneousCD30-positive T cell lymphoproliferative disorders, Primary cutaneousanaplastic large cell lymphoma, Lymphomatoid papulosis,Angioimmunoblastic T cell lymphoma, Peripheral T cell lymphoma(unspecified), Anaplastic large cell lymphoma), multiple myeloma (plasmacell myeloma or Kahler's disease).

Production of Cell-Binding Agent-Drug Conjugates

In order to link the cytotoxic compounds or derivative thereof of thepresent invention to the cell-binding agent, the cytotoxic compound maycomprise a linking moiety with a reactive group bonded thereto. In oneembodiment, a bifunctional crosslinking reagent can be first reactedwith the cytotoxic compound to provide the compound bearing a linkingmoiety with one reactive group bonded thereto (i.e., drug-linkercompound), which can then react with a cell binding agent.Alternatively, one end of the bifunctional crosslinking reagent canfirst react with the cell binding agent to provide the cell bindingagent bearing a linking moiety with one reactive group bonded thereto,which can then react with a cytotoxic compound. The linking moiety maycontain a chemical bond that allows for the release of the cytotoxicmoiety at a particular site. Suitable chemical bonds are well known inthe art and include disulfide bonds, thioether bonds, acid labile bonds,photolabile bonds, peptidase labile bonds and esterase labile bonds (seefor example U.S. Pat. Nos. 5,208,020; 5,475,092; 6,441,163; 6,716,821;6,913,748; 7,276,497; 7,276,499; 7,368,565; 7,388,026 and 7,414,073).Preferred are disulfide bonds, thioether and peptidase labile bonds.Other linkers that can be used in the present invention includenon-cleavable linkers, such as those described in are described indetail in U.S. publication number 2005/0169933, or charged linkers orhydrophilic linkers and are described in US 2009/0274713, US2010/01293140 and WO 2009/134976, each of which is expresslyincorporated herein by reference, each of which is expresslyincorporated herein by reference.

The compounds of formula (I)-(IV), (IA)-(IVA), and (IB)-(IVB) can belinked through R₁, R₂, R₃, R₄, R₁′, R₂′, R₃′, R₄′, L′, L″, L″, or X(when present). Of these, preferred linkable groups are R₂′, R₃′, R₄′,L′, L″, L′″ and most preferred linkable groups are R₂′, R₃′, and L′.Examples of linking groups for compounds of formula (I)-(IV),(IA)-(IVA), and (IB)-(IVB) are described above.

In one embodiment, a solution of an antibody in aqueous buffer may beincubated with a molar excess of an antibody modifying agent such asN-succinimidyl-3-(2-pyridyldithio)propionate (SPDP) or withN-succinimidyl-4-(2-pyridyldithio)butanoate (SPDB) to introducedithiopyridyl groups. The modified antibody is then reacted with thethiol-containing cytotoxic compound, such as compound 2a, to produce adisulfide-linked antibody-indolinobenzodiazepine dimer conjugate. Thecell binding agent-drug conjugate may then be purified using anypurification methods known in the art, such as those described in U.S.Pat. No. 7,811,572 and US Publication No. 2006/0182750, both of whichare incorporated herein by reference. For example, the cell-bindingagent-drug conjugate can be purified using tangential flow filtration,adsorptive chromatography, adsorptive filtration, selectiveprecipitation, non-absorptive filtration or combination thereof.Preferably, tangential flow filtration (TFF, also known as cross flowfiltration, ultrafiltration and diafiltration) and/or adsorptivechromatography resins are used for the purification of the conjugates.

Alternatively, the antibody may be incubated with a molar excess of anantibody modifying agent such as 2-iminothiolane, L-homocysteinethiolactone (or derivatives), or N-succinimidyl-5-acetylthioacetate(SATA) to introduce sulfhydryl groups. The modified antibody is thenreacted with the appropriate disulfide-containing cytotoxic agent, toproduce a disulfide-linked antibody-cytotoxic agent conjugate. Theantibody-cytotoxic agent conjugate may then be purified by methodsdescribed above. The cell binding may also be engineered to introducethiol moieties, such as cysteine-engineered antibodies disclosed in U.S.Pat. Nos. 7,772,485 and 7,855,275.

In another embodiment, a solution of an antibody in aqueous buffer maybe incubated with a molar excess of an antibody-modifying agent such asN-succinimidyl-4-(N-maleimidomethyl)-cyclohexane-1-carboxylate tointroduce maleimido groups, or withN-succinimidyl-4-(iodoacetyl)-aminobenzoate (SLAB) to introduceiodoacetyl groups. The modified antibody is then reacted with thethiol-containing cytotoxic agent to produce a thioether-linkedantibody-cytotoxic conjugate. The antibody-cytotoxic conjugate may thenbe purified by methods described above.

The number of cytotoxic molecules bound per antibody molecule can bedetermined spectrophotometrically by measuring the ratio of theabsorbance at 280 nm and 330 nm. An average of 1-10 cytotoxiccompounds/antibody molecule(s) can be linked by the methods describedherein. The preferred average number of linked cytotoxic compounds perantibody molecule is 2-5, and the most preferred is 2.5-4.0.

Cytotoxic agents containing linkers terminating in an N-hydroxysuccinimidyl (NHS) ester, such as compounds 1g and 10, can react withthe antibody to produce direct amide linked conjugates such ashuMy9-6-SPDB-1f or huMy9-6-BMPS-1f. The antibody-cytotoxic agentconjugate may then be purified by gel-filtration by any methodsdescribed above.

Representative processes for preparing the cell-binding agent-drugconjugates of the present invention are shown in FIGS. 22 and 23. Acytotoxic dimer compound of the present invention can be conjugated witha cell binding agent through either a one-step or a two-step conjugationmethod. In FIGS. 22 a and 22 b, representative examples are described,wherein a dimer compound that possesses a linker such as anN-hydroxysuccinimide ester is reacted directly with a cell bindingagent, such as an antibody, generating the desired conjugate. In FIG. 22c linkable dimer 1g was first treated with sodium bisulfate to provide amodified dimer compound 26 before adding antibody to form the conjugatehuMy9-6-SBDP-1f of the present invention.

A representative example of a two-step conjugation method is describedin FIG. 23, wherein an antibody is first modified with a bifunctionalcrosslinking agent resulting in an antibody that possesses a desirednumber of linkers suitable for reaction with a dimer compound having afree thiol moiety. In this example the antibody huMy9-6 was firstmodified with SPDB to give an antibody with linkers containing thedithiopyridyl moiety. The modified antibody was then exposed to a freethiol, such as 2a, generating the desired conjugate huMy9-6-SPDB-2a.

Processes for synthesizing the drug-linker compounds and conjugates ofthe invention are also described in U.S. provisional patent applicationNo. 61/443,092, filed on Feb. 15, 2011, and a U.S. utility applicationclaiming the benefit of filing date thereof and filed on the same day ofthe instant application, entitled “METHODS OF PREPARATION OFCONJUGATES,” the entire contents of which applications, including alldrawings, formulae, synthesis schemes, specifications, and claims, areincorporated herein by reference.

The structures of representative compounds and conjugates of the presentinvention are shown in Tables 1-8. These compounds and conjugates can beprepared according to the methods described herein.

TABLE 1 Structures of representative compounds in the present invention.

Notes: n = 1 or 3; m = 3 or 4 W = OH, OMe, ONHS, NHNH₂, H, Me, Ph,Peptide X = CH₂, O, S, NH or NMe Y = CH₂ or absent Z″ = H, Me, SMe,S(CH₂)₃C(O)NHS or CH₂C(O)NHS or BMPS or SMCC or SPy or SPy-NO₂

TABLE 2 Structures of representative compounds in the present invention(Continued).

Note: n = 1, 2 or 3 m = 3 or 4 W = OH, OMe, ONHS, NHNH₂, H, Me, Ph,Peptide X = CH₂, O, S, NH, NMe Y = absent or CH₂ Z = CH or N Z″ = H, Me,SMe, S(CH₂)₃C(O)NHS or CH₂C(O)NHS or BMPS or SMCC or SPy or SPy-NO₂

TABLE 3 Structures of representative compounds in the present invention(Continued).

Note: n = 1, 2 or 3 m = 3 or 4 W = OH, OMe, ONHS, NHNH₂, H, Me, Ph,Peptide X = CH₂, O, S, NH, NMe Z = CH or N Z″ = H, Me, SMe,S(CH₂)₃C(O)NHS or CH₂C(O)NHS or BMPS or SMCC or SPy or SPy-NO₂

TABLE 4 Structures of representative compounds in the present invention(Continued).

Note: n = 1, 2 or 3 m = 3 or 4 W = OH, OMe, ONHS, NHNH₂, H, Me, Ph,Peptide X = CH₂, O, S, NH, NMe Z = CH or N Z″ = H, Me, SMe,S(CH₂)₃C(O)NHS or CH₂C(O)NHS or BMPS or SMCC or SPy or SPy-NO₂

TABLE 5 Structures of representative compounds in the present invention.

TABLE 6 Structures of representative compounds in the present invention(Continued).

TABLE 7 Structures of representative compounds in the present invention(Continued).

TABLE 8 Structures of representative conjugates of the presentinvention.

In Vitro Cytotoxicity of Compounds and Conjugates

The cytotoxic compounds and cell-binding agent-drug conjugates of theinvention can be evaluated for their ability to suppress proliferationof various cancer cell lines in vitro. For example, cell lines such asthe human colon carcinoma line COLO 205, the rhabdomyosarcoma cell lineRH-30, and the multiple myeloma cell line MOLP-8 can be used for theassessment of cytotoxicity of these compounds and conjugates. Cells tobe evaluated can be exposed to the compounds or conjugates for 1-5 daysand the surviving fractions of cells measured in direct assays by knownmethods. IC₅₀ values can then be calculated from the results of theassays. Alternatively or in addition, an in vitro cell line sensitivityscreen, such as the one described by the U.S. National Cancer Institute(see Voskoglou-Nomikos et al., 2003, Clinical Cancer Res. 9: 42227-4239,incorporated herein by reference) can be used as one of the guides todetermine the types of cancers that may be sensitive to treatment withthe compounds or conjugates of the invention.

Examples of in vitro potency and target specificity ofantibody-cytotoxic agent conjugates of the present invention are shownin FIGS. 25-26. All of the conjugates are extremely cytotoxic on theantigen positive cancer cells with an IC₅₀ in the low picomolar range.Antigen negative cell lines remained viable when exposed to the sameconjugates. The indolinobenzodiazepine dimers showed target specificpotency being 160 fold less potent when blocked with unconjugatedantibody huMy9-6 (anti-CD33) and 40 less potent when blocked withunconjugated antibody FOLR1 (anti-folate receptor antibody). Forexample, the huMy9-6-SPDB-1f conjugate killed antigen positive HL60/QCcells with an IC₅₀ value of 10.5 pM, while the addition of an excess ofunconjugated huMy9-6 antibody reduced this cytotoxic effect (IC₅₀=1.69nM), demonstrating antigen specificity (FIG. 25A). In addition, thehuMy9-6-SPDB-1f conjugate is also highly potent towards both theHL60/ATCC cell line with an IC₅₀ value of 21 pM and the NB-4 cell linewith an IC₅₀ value of 190 pM (FIGS. 25B and 25C).

Similarly, the huFOLR1-SPDB-1f conjugate was highly potent, with an IC₅₀value of 55 pM for antigen positive KB cells (FIG. 26). Addition of anexcess of unconjugated huFOLR1 antibody reduced this cytotoxiceffect >40 fold, demonstrating antigen-specificity.

The effect of conjugation on antibody binding was measured by comparingthe binding of both unconjugated huMy9-6 antibody and thehuMy9-6-SPDB-1f conjugate towards the HL60/QC cell line (FIG. 27). FACSanalysis revealed that there is no change in binding capability of theconjugate to naked antibody indicating that there is no compromise inbinding due to conjugation of the cytotoxic agent to the antibody.

In one example, in vivo efficacy of a cell binding agent/cytotoxic agentconjugate was measured. Nude mice bearing human HL60/QC tumors weretreated with huMy9-6-SPDB-1f conjugate and significant tumor regressionwas observed at multiple doses while untreated mice grew tumors rapidly(FIG. 28). Activity was observed at doses as low as 20 μg/kg which is atleast 35-fold lower than the maximum tolerated dose.

The effect of imine saturation towards tolerability is shown in Table 9.Di-imine huFOLR1-drug1 was tested at multiple doses all of which werefound to be highly toxic leaving only survivors in the lowest grouptested at 50 μg/kg. In contrast the partially reduced mono-iminehuFOLR1-Drug 2 and huFOLR1-SPDB-IGN (huFOLR1-SPDB-1f) conjugates werefound to have significantly improved tolerability with thehuFOLR1-SPDB-IGN (huFOLR1-SPDB-1f) conjugate showing 100% animalsurvival at the highest doses tested of 560 μg/kg.

Compositions and Methods of Use

The present invention includes a composition (e.g., a pharmaceuticalcomposition) comprising novel benzodiazepine compounds described herein(e.g., indolinobenzodiazepine or oxazolidinobenzodiazepine), derivativesthereof, or conjugates thereof, (and/or solvates, hydrates and/or saltsthereof) and a carrier (a pharmaceutically acceptable carrier). Thepresent invention also includes a composition (e.g., a pharmaceuticalcomposition) comprising novel benzodiazepine compounds described herein,derivatives thereof, or conjugates thereof, (and/or solvates, hydratesand/or salts thereof) and a carrier (a pharmaceutically acceptablecarrier), further comprising a second therapeutic agent. The presentcompositions are useful for inhibiting abnormal cell growth or treatinga proliferative disorder in a mammal (e.g., human). The presentcompositions are also useful for treating depression, anxiety, stress,phobias, panic, dysphoria, psychiatric disorders, pain, and inflammatorydiseases in a mammal (e.g., human).

The present invention includes a method of inhibiting abnormal cellgrowth or treating a proliferative disorder in a mammal (e.g., human)comprising administering to said mammal a therapeutically effectiveamount of novel benzodiazepine compounds described herein (e.g.,indolinobenzodiazepine or oxazolidinobenzodiazepine), derivativesthereof, or conjugates thereof, (and/or solvates and salts thereof) or acomposition thereof, alone or in combination with a second therapeuticagent.

The present invention also provides methods of treatment comprisingadministering to a subject in need of treatment an effective amount ofany of the conjugates described above.

Similarly, the present invention provides a method for inducing celldeath in selected cell populations comprising contacting target cells ortissue containing target cells with an effective amount of a cytotoxicagent comprising any of the cytotoxic compound-cell-binding agents(e.g., indolinobenzodiazepine or oxazolidinobenzodiazepine dimer linkedto a cell binding agent) of the present invention, a salt or solvatethereof. The target cells are cells to which the cell-binding agent canbind.

If desired, other active agents, such as other anti-tumor agents, may beadministered along with the conjugate.

Suitable pharmaceutically acceptable carriers, diluents, and excipientsare well known and can be determined by those of ordinary skill in theart as the clinical situation warrants.

Examples of suitable carriers, diluents and/or excipients include: (1)Dulbecco's phosphate buffered saline, pH about 7.4, containing or notcontaining about 1 mg/mL to 25 mg/mL human serum albumin, (2) 0.9%saline (0.9% w/v NaCl), and (3) 5% (w/v) dextrose; and may also containan antioxidant such as tryptamine and a stabilizing agent such as Tween20.

The method for inducing cell death in selected cell populations can bepracticed in vitro, in vivo, or ex vivo.

Examples of in vitro uses include treatments of autologous bone marrowprior to their transplant into the same patient in order to killdiseased or malignant cells: treatments of bone marrow prior to theirtransplantation in order to kill competent T cells and preventgraft-versus-host-disease (GVHD); treatments of cell cultures in orderto kill all cells except for desired variants that do not express thetarget antigen; or to kill variants that express undesired antigen.

The conditions of non-clinical in vitro use are readily determined byone of ordinary skill in the art.

Examples of clinical ex vivo use are to remove tumor cells or lymphoidcells from bone marrow prior to autologous transplantation in cancertreatment or in treatment of autoimmune disease, or to remove T cellsand other lymphoid cells from autologous or allogenic bone marrow ortissue prior to transplant in order to prevent GVHD. Treatment can becarried out as follows. Bone marrow is harvested from the patient orother individual and then incubated in medium containing serum to whichis added the cytotoxic agent of the invention, concentrations range fromabout 10 μM to 1 pM, for about 30 minutes to about 48 hours at about 37°C. The exact conditions of concentration and time of incubation, i.e.,the dose, are readily determined by one of ordinary skill in the art.After incubation the bone marrow cells are washed with medium containingserum and returned to the patient intravenously according to knownmethods. In circumstances where the patient receives other treatmentsuch as a course of ablative chemotherapy or total-body irradiationbetween the time of harvest of the marrow and reinfusion of the treatedcells, the treated marrow cells are stored frozen in liquid nitrogenusing standard medical equipment.

For clinical in vivo use, the cytotoxic agent of the invention will besupplied as a solution or a lyophilized powder that are tested forsterility and for endotoxin levels. Examples of suitable protocols ofconjugate administration are as follows. Conjugates are given weekly for4 weeks as an intravenous bolus each week. Bolus doses are given in 50to 1000 mL of normal saline to which 5 to 10 mL of human serum albumincan be added. Dosages will be 10 μg to 2000 mg per administration,intravenously (range of 100 ng to 20 mg/kg per day). After four weeks oftreatment, the patient can continue to receive treatment on a weeklybasis. Specific clinical protocols with regard to route ofadministration, excipients, diluents, dosages, times, etc., can bedetermined by one of ordinary skill in the art as the clinical situationwarrants.

Examples of medical conditions that can be treated according to the invivo or ex vivo methods of inducing cell death in selected cellpopulations include malignancy of any type including, for example,cancer of the lung (small cell and non-small cell), breast, colon,brain, prostate, kidney, pancreas, ovary, head and neck, skin(melanoma), Merkel cell carcinoma, glioblastoma, neuroblastoma, andcancers of lymphatic organs; autoimmune diseases, such as systemiclupus, rheumatoid arthritis, and multiple sclerosis; graft rejections,such as renal transplant rejection, liver transplant rejection, lungtransplant rejection, cardiac transplant rejection, and bone marrowtransplant rejection; graft versus host disease; viral infections, suchas CMV infection, HIV infection, AIDS, etc.; and parasite infections,such as giardiasis, amoebiasis, schistosomiasis, and others asdetermined by one of ordinary skill in the art.

Cancer therapies and their dosages, routes of administration andrecommended usage are known in the art and have been described in suchliterature as the Physician's Desk Reference (PDR). The PDR disclosesdosages of the agents that have been used in treatment of variouscancers. The dosing regimen and dosages of these aforementionedchemotherapeutic drugs that are therapeutically effective will depend onthe particular cancer being treated, the extent of the disease and otherfactors familiar to the physician of skill in the art and can bedetermined by the physician. The contents of the PDR are expresslyincorporated herein in its entirety by reference. One of skill in theart can review the PDR, using one or more of the following parameters,to determine dosing regimen and dosages of the chemotherapeutic agentsand conjugates that can be used in accordance with the teachings of thisinvention. These parameters include:

Comprehensive index

By Manufacturer

Products (by company's or trademarked drug name)Category indexGeneric/chemical index (non-trademark common drug names)Color images of medicationsProduct information, consistent with FDA labelingChemical information

Function/action Indications & Contraindications

Trial research, side effects, warnings

Analogues and Derivatives

One skilled in the art of cytotoxic agents will readily understand thateach of the cytotoxic agents described herein can be modified in such amanner that the resulting compound still retains the specificity and/oractivity of the starting compound. The skilled artisan will alsounderstand that many of these compounds can be used in place of thecytotoxic agents described herein. Thus, the cytotoxic agents of thepresent invention include analogues and derivatives of the compoundsdescribed herein.

All references cited herein and in the examples that follow areexpressly incorporated by reference in their entireties.

EXAMPLES

The invention will now be illustrated by reference to non-limitingexamples. Unless otherwise stated, all percents, ratios, parts, etc. areby weight. All reagents were purchased from the Aldrich Chemical Co.,New Jersey, or other commercial sources. Nuclear Magnetic Resonance (¹HNMR) spectra were acquired on a Bruker 400 MHz instrument and massspectra were acquired on a Bruker Daltonics Esquire 3000 instrumentusing electrospray ionization.

Example 1

Compound 1b:

To a stirred solution of the aniline 1a (1.55 g, 5.18 mmol) and2-(methyldithio)-isobutyraldehyde (0.7 mL, 5.18 mmol) in anhydrous1,2-dichloromethane (20 mL) was added sodium triacetoxyborohydride (1.1g, 5.18 mmol) and zinc chloride powder (353 mg, 2.59 mmol) followed bythe addition of anhydrous magnesium sulfate (800 mg). The mixture wasstirred at room temperature (rt) for 6 hours then a second portion of2-(methyldithio)-isobutyraldehyde (0.7 mL, 5.18 mmol) and sodiumtriacetoxyborohydride (1.1 g, 5.18 mmol) were added. It continued to bestirred at rt overnight. The reaction mixture was filtered throughcelite and washed with dichloromethane. The filtrate was concentratedand the remainder was purified by silica gel chromatography (Combiflash,40 g column, dichloromethane/MeOH) to give compound 1b (487 mg y=22%) ascolorless oil. Unreacted starting material aniline 1a (1.02 g) was alsorecovered in 65% yield. ¹H NMR (400 Hz, CDCl₃): δ 6.76 (s, 2H), 6.63 (s,1H), 4.55 (s, 4H), 3.65-3.51 (m, 14H), 3.35 (s, 3H), 2.44 (s, 3H), 1.33(s, 6H); ¹³C NMR (400 Hz, CDCl₃): δ 149.0, 142.35, 114.0, 111.1, 71.98,70.7, 70.6, 70.5, 67.6, 65.5, 59.75, 59.1, 53.9, 51.9, 26.6, 25.7,20.75; MS (m/z): found 456.2 (M+Na)⁺. See FIG. 1.

Compound 1c:

To a stirred solution of 1b (243 mg, 0.56 mmol) in anhydrousdichloromethane (3.5 mL) was added triethylamine (234 μl, 1.68 mmol).The mixture was cooled to −10° C. and methanesulfonyl chloride (113 μl,1.46 mmol) was added slowly over 15 minutes via a syringe. The solutioncontinued to be stirred for 60 minutes at −10 ˜−7° C. and quenched byaddition of ice/water. It was diluted with ethyl acetate and washed withcold water. The organic layer was dried over anhydrous sodium sulfate,filtered, concentrated and high vacuumed to give the mesylates as lightyellowish oil (340 mg). The mesylates was transferred into a 10 mLround-bottomed flask with ethyl acetate/dichloromethane, concentratedand high vacuumed. IBD monomer (412 mg, 1.4 mmol) was added followed bythe addition of anhydrous dimethylformamide (3 mL) and anhydrouspotassium carbonate (232 mg, 1.68 mmol). The obtained yellowish mixturewas stirred at room temperature overnight. It was diluted withdichloromethane and washed with brine. The organic layer was dried overanhydrous sodium sulfate, filtered and concentrated. The residue wasdissolved in dichloromethane and loaded on silica gel column and elutedwith dichloromethane/methanol (15:1 then 10:1). The fractions thatcontained compound 1c were combined and concentrated to give 705 mg ofcrude product which was further purified by preparative reverse phaseHPLC (C18 column, eluted with acetonitrile/water) to give compound 1c asa yellowish fluffy solid (181 mg, y=33%). ¹H NMR (400 Hz, CDCl₃): δ 8.28(d, J=8.0 Hz, 2H), 7.86 (d, J=3.6 Hz, 2H), 7.59 (s, 2H), 7.31-7.26 (m,4H), 7.12 (t, J=7.6 Hz, 2H), 6.87-6.80 (m, 5H), 5.18 (dd, J₁=20.8 Hz,J₂=12.4 Hz, 4H), 4.50-4.47 (m, 2H), 3.99 (s, 6H), 3.75-3.48 (m, 18H),3.37 (s, 3H), 2.44 (s, 3H), 1.32 (s, 6H); MS (m/z): found 1025.9(M+H₂O+Na)⁺, 1043.9 (M+2H₂O+Na)⁺, 983.8 (M−H)⁻, 1055.8 (M+4H₂O—H)⁻. SeeFIG. 1.

Compound 1d:

To a stirred solution of compound 1c (112 mg, 0.114 mmol) in anhydrousdichloromethane (0.3 mL) and absolute ethanol (0.6 mL) was added sodiumborohydride (0.9 mg, 0.023 mmol) at 0° C. The ice bath was removed after5 minutes and the mixture was stirred at room temperature for 3 hoursand then cooled to 0° C. and quenched with saturated ammonium chloride,diluted with dichloromethane, separated and the organic layer was washedwith brine, dried over anhydrous Na₂SO₄ and filtered through celite andconcentrated. The residue was purified by reverse phase HPLC (C18column, acetonitrile/water). The corresponding fractions were extractedwith dichloromethane and concentrated to obtain the products 1d, 1e andthe unreacted starting material 1c. Compound 1d: 37.1 mg (y=33%), MS(m/z): found 1010.4 (M+Na)⁺, 1028.4 (M+H₂O+Na)⁺, 1040.3 (M+3H₂O—H)⁻;compound 1e: 6.4 mg (y=5.7%), MS (m/z): found 1012.4 (M+Na)⁺; compound1c: 44.1 mg (y=39%). See FIG. 1.

Compound 1g:

To a stirred solution of 1d (23.6 mg, 0.024 mmol) in acetonitrile (3 mL)and methanol (3 mL) was added freshly prepared TCEP solution (17 mg ofTCEP HCl salt was neutralized with saturated sodium bicarbonate to pH6-6.5 then diluted with 0.5 mL of pH 6.5 phosphate buffer) at roomtemperature. The mixture was stirred at room temperature for 3 hours andthen diluted with dichloromethane and deionized water, separated and theorganic layer was washed with brine, dried over anhydrous sodium sulfateand filtered. The filtrate was concentrated and high vacuumed to give 22mg of if as light yellowish foam. Another 18 mg of if was prepared from19 mg of 1d following the same procedure. The combined 40 mg (0.042mmol) of if was dissolved in anhydrous dichloromethane (0.5 mL) andstirred. To this stirred solution was added SPDB NHS ester 2 (34.6 mg,80% purity, 0.085 mmol) and diisopropylethylamine (15 μl, 0.085 mmol).It continued to be stirred at room temperature overnight, quenched withsaturated ammonium chloride and diluted with dichloromethane, separatedand washed with brine, dried over sodium sulfate, filtered andconcentrated. The residue was purified by preparative reverse phase HPLC(C18 column, acetonitrile/water). The fractions containing product werecombined, extracted with dichloromethane and concentrated to givecompound 1g as white solid (29.7 mg, y=60%). ¹H NMR (400 Hz, CD₃CN): δ8.28-8.25 (m, 1H), 8.20-8.17 (m, 1H), 7.87-7.84 (m, 1H), 7.49 (d, J=4.4Hz, 1H), 7.39 (d, J=4.4 Hz, 1H), 7.31-7.19 (m, 4H), 7.13-7.01 (m, 2H),6.92-6.87 (m, 3H), 6.77 (bs, 1H), 6.31-6.29 (m, 1H), 5.16-5.09 (m, 2H),5.00 (d, J=4.4 Hz, 2H), 4.94 (bs, —NH), 4.48-4.43 (m, 1H), 4.40-4.34 (m,1H), 3.90 (d, J=4.4 Hz, 3H), 3.77 (d, J=4.4 Hz, 3H), 3.64-3.39 (m, 18H),3.26 (d, J=4.4 Hz, 3H), 2.82-2.70 (m, 8H), 2.17 (d, J=4.4 Hz, 1H),2.08-2.01 (m, 3H), 1.30 (d, J=4.4 Hz, 6H); MS (m/z): found 1025.9(M+H₂O+Na)⁺, 1043.9 (M+2H₂O+Na)hu +, 983.8 (M−H)⁻, 1055.8 (M+4H₂O−H)⁻;MS (m/z), found 1179.5 (M+Na)⁺. See FIG. 1.

Example 2

Compound 1e:

To a stirred solution of 1c (8 mg, 0.0081 mmol) in anhydrous1,2-dichloromethane (0.2 mL) was added sodium triacetoxyborohydride (3.8mg, 0.018 mmol). The mixture was stirred at room temperature for 1.5hours, then the mixture was diluted with dichloromethane and quenchedwith saturated sodium bicarbonate, separated and the organic layer waswashed with brine, dried over sodium sulfate, and filtered. The filtratewas concentrated and the remainder was purified by reverse phase HPLC(C18 column, acetonitrile/water) to give compound 1e as a white solid(4.7 mg, y=58%). MS (m/z), found 1012.4 (M+Na)⁺, 1024.2 (M+2H₂O−H)⁻. SeeFIG. 2.

Compound 2a:

To a stirred solution of compound 1e (12 mg, 0.012 mmol) in acetonitrile(1 mL) and methanol (3 mL) was added freshly prepared TCEP solution (11mg of TCEP HCl salt was neutralized with saturated sodium bicarbonate topH ˜6.5 then diluted with 0.4 mL of pH 6.5 phosphate buffer) at roomtemperature. The mixture was stirred at room temperature for 3.5 hoursand then diluted with dichloromethane and deionized water, separated andthe organic layer was washed with brine, dried over anhydrous sodiumsulfate and filtered. The filtrate was concentrated and the remainderwas purified by reverse phase HPLC (C18 column, acetonitrile/water) togive compound 2a as a white solid (4.9 mg, y=43%). MS (m/z), found 966.4(M+Na)⁺, 978.2 (M+2H₂O−H)⁻. See FIG. 2.

Example 3

Compound 3b:

To a solution of compound 3a (830 mg, 1.9 mmol) in methanol (15 mL) wasadded Pd/C (10%, 204 mg, 0.19 mmol). The air in the flask was removed byvacuum and then replaced with hydrogen in a balloon. The mixture wasstirred at room temperature overnight. The mixture was filtered throughcelite and washed the celite/Pd/C with dichloromethane and methanol. Thefiltrate was concentrated and the residue diluted with dichloromethaneand evaporated for a few cycles and then was purified by silica gelchromatography (dichloromethane/methanol) to give compound 3b as a lightyellowish solid (558 mg, y=98%). ¹H NMR (400 Hz, CDCl₃): δ 8.34 (d,J=8.0 Hz, 1H), 7.40 (s, 1H), 7.22 (dd, J₁=8.0 Hz, J₂=7.6 Hz, 1H), 7.17(d, J=7.2 Hz, 1H), 7.02 (dd, J₁=7.2 Hz, J₂=7.6 Hz, 1H), 6.16 (s, 1H),4.37 (tt, J₁=10.4 Hz, J₂=7.2 Hz, 1H), 3.76 (s, 3H), 3.49-3.36 (m, 3H),2.73 (dd, J₁=16.8 Hz, J₂=3.6 Hz, 1H); ¹³C NMR (400 Hz, CDCl₃): δ 167.0,150.4, 142.6, 141.2, 140.8, 129.9, 127.7, 124.8, 123.96, 117.4, 113.7,112.5, 104.7, 57.3, 56.3, 54.7, 33.0; MS (m/z), found 295.1 (M−H)⁻. SeeFIG. 3.

Compound 3c:

To a solution of the 2-(methyldithio)-isobutyraldehyde (113 mg, 0.75mmol) and compound 3b (148 mg, 0.5 mmol) in anhydrous 1,2-dichloroethane(2 mL) was added sodium triacetoxyborohydride (212 mg, 1.0 mmol). Themixture was stirred at room temperature for 2 days. During the time,another two portions (0.05 mL, 0.5 mmol/portion) of2-(methyldithio)-isobutyraldehyde along with one portion of sodiumtriacetoxyborohydride (106 mg, 0.5 mmol) were added. The reaction wasquenched with saturated sodium bicarbonate, diluted with dichloromethaneand water. The organic layer was washed with brine, dried over sodiumsulfate and filtered. The filtrate was concentrated under reducedpressure and the residue was purified by silica gel chromatography(Combiflash, 24 g column, hexanes/ethyl acetate) to give compound 3c asa white fluffy solid (92.5 mg, y=43%). Unreacted starting material 3bwas also recovered (49.3 mg, y=33%). ¹H NMR (400 Hz, CDCl₃): δ 8.30 (d,J=8.0 Hz, 1H), 7.28 (dd, J₁=6.8 Hz, J₂=7.6 Hz, 1H), 7.25-7.20 (m, 2H),7.07 (t, J=7.6 Hz, 1H), 6.80 (s, 1H), 6.17 (s, 1H), 4.36-4.28 (m, 1H),3.89 (s, 3H), 3.78 (d, J=14.4 Hz, 1H), 3.46-3.34 (m, 3H), 2.90 (d,J=14.4 Hz, 1H), 2.73 (dd, J₁=16.4 Hz, J₂=2.8 Hz, 1H), 2.34 (s, 3H), 1.17(s, 3H), 1.05 (s, 3H); ¹³C NMR (400 Hz, CDCl₃): δ 167.2, 149.0, 142.5,142.2, 141.9, 129.9, 128.0, 125.3, 124.5, 124.1, 117.1, 112.0, 108.5,64.8, 61.4, 58.1, 56.3, 53.4, 32.0, 26.3, 25.7, 25.4; MS (m/z), found453.3 (M+Na)⁺, 429.2 (M−H)⁻. See FIG. 3.

Compound 3d:

To a stirred solution of IBD monomer (125 mg, 0.425 mmol) and1,5-diiodopentane (0.63 mL, 4.25 mmol) in anhydrous dimethylformamide (3mL) was added potassium carbonate (59 mg, 0.425 mmol) and the mixturewas stirred at room temperature overnight. The reaction solution wasdiluted with dichloromethane, washed with brine and dried over anhydroussodium sulfate. It was filtered and concentrated. The residue waspurified by silica gel chromatography (hexanes/ethyl acetate) to givecompound 3d as yellowish foam (94 mg, y=45%). ¹H NMR (400 Hz, CDCl₃): δ8.27 (d, J=8.0 Hz, 1H), 7.86 (d, J=4.8 Hz, 1H), 7.56 (s, 1H), 7.27 (dd,J₁=8.4 Hz, J₂=7.6 Hz, 2H), 7.10 (dd, J₁=7.6 Hz, J₂=7.2 Hz, 1H), 6.82 (s,1H), 4.48 (dt, b=10.8 Hz, J₂=4.4 Hz, 1H), 4.15-4.07 (m, 2H), 3.96 (s,3H), 3.70 (dd, J₁=16.8 Hz, J₂=10.8 Hz, 1H), 3.49 (dd, J₁=16.8 Hz, J₂=4.0Hz, 1H), 3.22 (t, J=7.2 Hz, 2H), 1.96-1.87 (m, 4H), 1.64-1.57 (m, 2H);¹³C NMR (400 Hz, CDCl₃): δ 164.0, 163.2, 151.4, 148.3, 142.2, 140.3,129.6, 128.3, 124.9, 120.5, 117.0, 112.0, 110.6, 68.8, 56.4, 55.1, 33.3,32.7, 28.0, 27.2, 6.6; MS (m/z), found 513.3 (M+Na)⁺, 543.2 (M+3H₂O−H)⁻.See FIG. 3.

Compound 3e:

To a stirred solution of the starting materials 3c (91 mg, 0.21 mmol)and 3d (94 mg, 0.19 mmol) in anhydrous dimethylformamide (1 mL) wasadded potassium carbonate (29 mg, 0.21 mmol) and the mixture was stirredat room temperature overnight. The reaction solution was diluted withdichloromethane, washed with brine and dried over anhydrous sodiumsulfate. It was filtered, concentrated and the residue was purified bysilica gel chromatography (hexanes/ethyl acetate) to give compound 3e asyellowish foam (89.1 mg, y=58%). ¹H NMR (400 Hz, CDCl₃): δ 8.32-8.28 (m,2H), 7.91 (bs, 1H), 7.57 (s, 1H), 7.36-7.21 (m, 5H), 7.15-7.05 (m, 2H),6.85 (s, 1H), 6.74 (s, 1H), 4.53-4.48 (m, 1H), 4.37-4.31 (m, 1H),4.21-4.03 (m, 4H), 3.98 (s, 3H), 3.88 (s, 3H), 3.86-3.70 (m, 2H),3.55-3.35 (m, 4H), 2.93 (d, J=4.0 Hz, 1H), 2.73 (dd, b=16.4 Hz, J₂=2.4Hz, 1H), 2.36 (s, 3H), 2.03-1.96 (m, 3H), 1.77-1.67 (m, 3H), 1.21 (s,3H), 1.06 (s, 3H); MS (m/z), found 815.3 (M+Na)⁺. See FIG. 3.

Compound 3g:

To a stirred solution of compound 3e (33.1 mg, 0.042 mmol) inacetonitrile (2 mL) and methanol (4 mL) was added freshly prepared TCEPsolution (36 mg of TCEP HCl salt was neutralized with saturated sodiumbicarbonate to pH ˜6.5 then diluted with 0.4 mL of pH 6.5 phosphatebuffer) at room temperature. The mixture was stirred at room temperaturefor 3 hours and then diluted with dichloromethane and deionized water,separated and the organic layer was washed with brine, dried overanhydrous sodium sulfate and filtered. The filtrate was concentrated andhigh vacuumed to give 31 mg of compound 3f as yellowish solid. It wasdissolved in anhydrous dichloromethane (0.5 mL). SPDB NHS ester 2 (26mg, 80% purity, 0.063 mmol) and diisopropylethylamine (11 μl, 0.063mmol) were added subsequently. The mixture continued to be stirred atroom temperature overnight, quenched with saturated ammonium chlorideand diluted with dichloromethane, separated and washed with brine, driedover sodium sulfate, filtered and concentrated. The residue was purifiedby preparative reverse phase HPLC (C18 column, acetonitrile/water). Thefractions containing product were combined, extracted withdichloromethane and concentrated to give compound 3g as yellowish solid(15.2 mg, y=38%). MS (m/z), found 984.3 (M+Na)⁺, 1014.2 (M+3H₂O−H)⁻. SeeFIG. 3.

Example 4

Compound 4b:

A stirred solution of compound 4a (111 mg, 0.108 mmol) in absoluteethanol (720 μL) and anhydrous dichloromethane (360 μL) was cooled to 0°C. in an ice bath. Sodium borohydride (0.817 mg, 0.022 mmol) in 50 uLabsolute ethanol was added at 0° C. The reaction stirred at ambienttemperature for two hours. The mixture was cooled to 0° C. in an icebath, quenched with saturated ammonium chloride and extracted withdichloromethane. The organic extracts were washed with brine, dried overanhydrous sodium sulfate and filtered through Celite. The filtrate wasconcentrated under reduced pressure and the crude material was purifiedby RP-HPLC (C18 DI water/acetonitrile) to yield compound 4b (43 mg,38%). ¹H NMR (400 Hz, CDCl₃): δ8.26 (d, 1H, J=8.0 Hz), 8.18 (d, 1H,J=8.0 Hz), 7.77 (d, 1H, J=4.4 Hz), 7.51 (s, 1H), 7.41 (s, 2H), 7.17 (m,6H), 7.03 (t, 1H, J=7.2 Hz), 6.96 (t, 1H, J=7.2 Hz), 6.76 (s, 1H), 6.04(s, 1H), 5.13 (m, 4H), 4.38 (m, 2H), 3.90 (s, 3H), 3.81 (s, 3H), 3.79(m, 2H), 3.63 (m, 1H), 3.51 (m, 8H), 3.43 (m, 6H), 3.25 (s, 3H), 2.73(dd, 1H, J=3.6, 16.4 Hz), 2.22 (s, 3H), 2.04 (m, 2H), 1.81 (m, 2H), 1.18(s, 6H); MS (m/z) found, 1051.9 (M+Na), 1069.9 (M+Na⁺H₂O). See FIG. 4.

Compound 4c:

To a stirred solution of compound 4b (40 mg, 0.039 mmol) in methanol(4.45 mL) and acetonitrile (2.225 mL) was added TCEP.HCl (39.0 mg, 0.136mmol) in sodium phosphate buffer (0.89 mL, pH 6.5). The mixture stirredat ambient temperature for 18 hours. The mixture was diluted withdichloromethane and washed with water and brine. The organic extractswere dried over anhydrous sodium sulfate, filtered and concentrated.Purification by RP-hPLC (C18, DI water/acetonitrile) and extraction withdichloromethane yielded compound 4c (26.5 mg, 64%); MS (m/z) found,1006.0 (M+Na). See FIG. 4.

Compound 4d:

To a stirred solution of compound 4c (24 mg, 0.024 mmol) in anhydrousdichloromethane (800 μL) was added PBA (11.18 mg, 0.049 mmol) anddiisopropylethylamine (20.18 μL, 0.116 mmol). After stirring for 18hours at ambient temperature the reaction was diluted withdichloromethane and quenched with saturated ammonium chloride. Thelayers were separated and the organic was washed with brine, dried overanhydrous sodium sulfate, filtered and concentrated under reducedpressure. Purification by PTLC (5% methanol/dichloromethane) yieldedcompound 4d (17 mg, 63%); MS (m/z) found, 1123.9 (M+Na) 1139.9 (M+K);1099.8 (M−H) 117.9 (M−H+H₂O). See FIG. 4.

Compound 4e:

To a mixture of compound 4d (15 mg, 0.014 mmol) and N-hydroxysuccinimide (4.70 mg, 0.041 mmol) in anhydrous dichloromethane (1.0 mL)was added EDC.HCl (7.83 mg, 0.041 mmol). After stirring 18 hours atambient temperature the reaction was diluted with dichloromethane andquenched with saturated ammonium chloride. The mixture was washed withbrine, dried over anhydrous sodium sulfate, filtered and concentratedunder reduced pressure. The crude material was purified by RP-HPLC (C18,DI water/acetonitrile). Fractions containing product were pooled andextracted with dichloromethane, dried over anhydrous magnesium sulfate,filtered and concentrated to give compound 4e (13 mg, 80%); MS (m/z)found, 1220.8 (M+Na) 1238.8 (M+Na⁺H₂O), 1254.8 (M+K+H₂O). See FIG. 4.

Example 5

Compound 5a:

A mixture of chelidamic acid hydrate (3.0 g, 15.56 mmol) and sulfuricacid (0.6 mL, 11.26 mmol) in absolute ethanol (40 mL) was refluxed for20 hours. The reaction was cooled to ambient temperature, neutralizedwith aqueous sodium carbonate, and then acidified with concentrated HCl.Water was added and the mixture was extracted with dichloromethane. Theextracts were dried with anhydrous magnesium sulfate, filtered andconcentrated. The crude material was purified by silica gelchromatography (5% methanol/dichloromethane) to yield diethyl4-hydroxypyridine-2,6-dicarboxylate (5a) (2.5 g, 68%) as a white solid.See FIG. 5.

Compound 5c:

A solution of 4-methyl-4-(methyldisulfanyl)pentan-1-ol (5b) (2.0 g,11.09 mmol) in anhydrous dichloromethane (55.5 mL) was cooled to 0° C.in an ice bath. Triethylamine (5.41 mL, 38.8 mmol) and toluene sulfonylchloride (3.17 g, 16.64 mmol) were added at 0° C. The reaction stirredfor three hours at ambient temperature. The mixture was extracted withethyl acetate and washed with brine. The organic extracts were driedwith anhydrous sodium sulfate, filtered and concentrated. Purificationby silica gel chromatography (5% ethyl acetate/hexanes) resulted in4-methyl-4-(methyldisulfanyl)pentyl 4-methylbenzenesulfonate (5c) (1.5g, 40%). 5b: ¹H NMR (400 Hz, CDCl₃): δ3.42 (m, 2H), 2.19 (s, 3H), 1.77(bs, 1H), 1.43 (m, 4H), 1.09 (s, 6H). 5c: ¹H NMR (400 Hz, CDCl₃): δ7.66(d, 2H, J=7.6 Hz), 7.22 (d, 2H, J=8.0 Hz), 3.90 (t, 2H, J=6.4 Hz), 2.32(s, 3H), 2.23 (s, 3H), 1.60 (m, 2H), 1.44 (m, 2H), 1.11 (s, 6H). SeeFIG. 5.

Compound 5d:

To a stirred solution of 4-methyl-4-(methyldisulfanyl)pentyl4-methylbenzenesulfonate (5c) (0.48 g, 1.435 mmol) and diethyl4-hydroxypyridine-2,6-dicarboxylate (5a)(0.343 g, 1.435 mmol) inanhydrous dimethylformamide (6.5 mL) was added Potassium carbonate(0.297 g, 2.152 mmol). The reaction was stirred at 90° C. for 18 hours.Then allowed to cool to ambient temperature and quenched with saturatedammonium chloride. The mixture was extracted three times with ethylacetate. The extracts were dried with anhydrous sodium sulfate, filteredand concentrated under reduced pressure. Purification by silica gelchromatography (30% hexanes/ethyl acetate) yielded diethyl4-(4-methyl-4-(methyldisulfanyl)pentyloxy)pyridine-2,6-dicarboxylate(5d)(300 mg, 52%); ¹H NMR (400 Hz, CDCl₃): δ 7.70 (s, 2H), 4.40 (q, 4H,J=7.2, 14.4 Hz), 4.07 (t, 2H, J=6. Hz), 2.35 (s, 3H), 1.86 (m, 2H), 1.70(m, 2H), 1.38 (t, 6H, J=7.2 Hz), 1.27 (s, 6H); MS (m/z), found 424.1(M+Na), 440.1 (M+K). See FIG. 5.

Compound 5e:

To a stirred solution of diethyl4-(4-methyl-4-(methyldisulfanyl)pentyloxy)pyridine-2,6-dicarboxylate(5d) (270 mg, 0.672 mmol) in absolute ethanol (7.0 mL) was added calciumchloride (224 mg, 2.017 mmol) and sodium borohydride (76 mg, 2.017mmol). The reaction was allowed to stir at ambient temperature for 90minutes after which it was quenched with water and concentrated in vacuoto remove the ethanol. The mixture was then extracted twice withdichloromethane. The organic extracts were combined, washed with water,dried with anhydrous magnesium sulfate and filtered through celite. Thefiltrate was concentrated under reduced pressure and the crude materialwas purified by silica gel chromatography eluting with 10%methanol/dichloromethane to yield(4-(4-methyl-4-(methyldisulfanyl)pentyloxy)pyridine-2,6-diyl)dimethanol(5e)(75 mg, 35%); ¹H NMR (400 Hz, CDCl₃): δ 6.63 (s, 2H), 4.60 (s, 4H),3.95 (t, 2H, J=6.2 Hz), 3.54 (bs, 2H), 2.35 (s, 3H), 1.82 (m, 2H), 1.66(m, 2H), 1.26 (s, 6H); MS (m/z), found 340.1 (M+Na). See FIG. 5.

Compound 5f:

A stirred solution of(4-(4-methyl-4-(methyldisulfanyl)pentyloxy)pyridine-2,6-diyl)dimethanol(5e) (51 mg, 0.161 mmol) in anhydrous dichloromethane (1.6 mL) wascooled to −5° C. in an acetone/ice bath. Triethylamine (0.112 mL, 0.803mmol) and methanesulfonyl chloride (0.031 mL, 0.402 mmol) were added.The mixture was stirred for 60 minutes at −5° C. The reaction wasquenched with ice water and extracted with cold ethyl acetate. Theorganic extracts were washed with ice water, dried with anhydrous sodiumsulfate, filtered and concentrated under reduced pressure to give thedimesylate. To a stirred mixture of the dimesylate intermediate (179 mg,0.378 mmol) and IBD monomer (256 mg, 0.869 mmol) in anhydrousdimethylformamide (3.8 mL) was added potassium carbonate (261 mg, 1.890mmol) and potassium iodide (31.4 mg, 0.189 mmol). The reaction wasallowed to stir at ambient temperature for 18 hours. The mixture wasquenched with water and extracted three times with dichloromethane. Theorganic extracts were dried with sodium sulfate, filtered andconcentrated. The crude material was redissolved in acetonitrile andpurified by RP-HPLC (C18, deionized water/acetonitrile). Fractionscontaining product were combined and extracted with dichloromethane,dried with anhydrous magnesium sulfate, filtered and concentrated underreduced pressure to yield compound 5f (65 mg, 20%); ¹H NMR (400 Hz,CDCl₃): δ 8.20 (d, 2H, J=8.0 Hz), 7.78 (m, 2H), 7.53 (s, 2H), 7.20 (m,4H), 7.04 (t, 2H, J=7.4 Hz), 6.91 (m, 2H), 6.80 (s, 2H), 5.22 (s, 4H),4.40 (m, 2H), 3.94 (s, 6H), 3.93 (m, 2H), 3.63 (m, 2H), 3.42 (dd, 2H,J=Hz), 2.32 (s, 3H), 1.80 (m, 2H), 1.64 (m, 2H), 1.24 (s, 6H); MS (m/z),found 892.3 (M+Na) 910.3 (M+Na+H₂O) 928.3 (M+Na+2H₂O). See FIG. 5.

Compound 5g and 5h:

A solution of compound 5f (74 mg, 0.085 mmol) in absolute ethanol (600μL) and anhydrous dichloromethane (300 μL) was cooled 0° C. in an icebath. Sodium borohydride (0.644 mg, 0.017 mmol) in 50 μL absoluteethanol was added at 0° C. The mixture was allowed to stir at ambienttemperature for two hours and was then cooled to 0° C. The reaction wasquenched with saturated ammonium chloride and extracted withdichloromethane. The organic extracts were washed with brine, dried overanhydrous sodium sulfate, filtered through Celite and concentrated underreduced pressure. The crude material was redissolved indimethylformamide and purified by RP-HPLC (C18 deionizedwater/acetonitrile). Fractions containing compounds 5g and 5h werecombined separately and extracted with dichloromethane, dried withanhydrous magnesium sulfate, filtered and concentrated to yield compound5g (20 mg, 27%) and compound 5h. 5g: ¹H NMR (400 Hz, CDCl₃): δ 8.25 (m,1H), 8.18 (m, 1H), 7.77 (m, 1H), 7.51 (ss, 1H), 7.40 (ss, 1H), 7.18 (m,4H), 7.08 (m, 1H), 7.03 (m, 1H), 6.92 (m, 2H), 6.86 (ss, 1H) 5.98/6.06(ss, 1H), 5.24 (m, 4H), 4.40 (m, 1H), 4.30 (m, 1H), 3.94 (s, 3H), 3.92(m, 2H), 3.84 (s, 3H), 3.62 (m, 1H), 3.37 (m, 4H), 2.65 (m or dd, 1H),2.32 (ss, 3H), 1.77 (m, 2H), 1.64 (m, 2H), 1.24 (s, 6H). 5h: ¹H NMR (400Hz, CDCl₃): δ 8.24 (d, 2H, J=8.0 Hz), 7.39 (s, 2H), 7.14 (m, 4H), 6.97(m, 2H), 6.93 (m, 2H), 6.15 (ss, 2H), 5.25 (s, 4H), 4.37 (m or t, 2H,J=9.8 Hz), 4.2 (bs, 2H), 3.94 (m, 2H), 3.83 (s, 6H), 3.40 (m, 6H), 2.72(dd, 2H, J=Hz), 2.32 (s, 3H), 1.79 (m, 2H), 1.64 (m, 2H), 1.24 (s, 6H).See FIG. 5.

Compound 5i:

To a stirred solution of compound 5g (20 mg, 0.023 mmol) in methanol(5.25 mL) and acetonitrile (1.750 mL) was added TCEP.HCl (19.72 mg,0.069 mmol) in sodium phosphate buffer (0.7 mL, pH 6.5). The mixture wasstirred for 3 hours at ambient temperature and then diluted withdichloromethane and water. The layers were separated and the organic waswashed with brine. The crude product was purified by RP-HPLC (C18,deionized water/acetonitrile). Fractions containing product werecombined, extracted with dichloromethane and evaporated to yieldcompound 5i (7 mg, 37%). MS (m/z), found 848.3 (M+Na) 866.3 (M+Na+H₂O)880.3 (M+Na⁺ MeOH). See FIG. 5.

Compound 5j:

To a stirred solution of compound 5i (7 mg, 8.47 μmol) and2,5-dioxopyrrolidin-1-yl 4-(pyridin-2-yldisulfanyl)butanoate (8.64 mg,0.021 mmol) in anhydrous dichloromethane (113 μL) was addeddiisopropylethylamine (3.69 μL, 0.021 mmol). After stirring for 18 hoursat ambient temperature the reaction was quenched with saturated ammoniumchloride solution and extracted with dichloromethane. The organicextracts were washed with brine, dried with anhydrous sodium sulfate,filtered and concentrated under reduced pressure. The crude material waspurified by preparative RP-HPLC (C18, deionized water/acetonitrile).Fractions containing product were extracted with dichloromethane,filtered and evaporated to yield compound 5j (3 mg, 34%). MS (m/z),found 1063.3 (M+Na) 1081.3 (M+Na+H₂O). See FIG. 5.

Example 6 Preparation of Antibody-SPDB-Drug Conjugate

Compound 1g was pre-treated with 3 molar equivalents of sodium bisulfite(using a freshly prepared NaHSO₃ solution in water) in 96-98% DMA inwater for 4-5 hrs at 25° C. For conjugation, the humanized antibody at 2mg/mL was reacted with 5-7 molar equivalents of compound 1g (pre-treatedwith NaHSO₃) for 6 h at 25° C. in 85-90% PBS, pH 7.4, aqueous buffer, or50 mM HEPES, pH 8.5, aqueous buffer, containing 10-15%N,N-dimethylacetamide (DMA) and then purified over a G25 gel filtrationcolumn in PBS, pH 7.4, to remove unreacted or hydrolyzed drug compound.The humanized antibody-SPDB-drug conjugates were dialyzed in 10 mMHistidine, 250 mM Glycine, 1% sucrose, pH 6.5 buffer. The Drug AntibodyRatio (DAR) of the conjugates were measured to be 2.2-2.9 by UVabsorbance measurements at 280 and 320 nm and using the extinctioncoefficients of the drug and antibody at 280 nm (215,000 M⁻¹cm⁻¹) and320 nm (9137 M⁻¹cm⁻¹). The percentage of monomer in the conjugates weredetermined as >90% by SEC (Size Exclusion Chromatography) using TSK-GelG300SWXL column (7.8 mm×300 mm, 5 μm particle size). Based on the UVabsorbance of the monomer peak in SEC it was also demonstrated that themonomer conjugate peaks had linked drug molecules. For free(unconjugated) drug assay, the conjugate was acetone extracted to removeprotein, dried, and reconstituted in mobile phase and injected onto aVYDAC 208TP C8 reverse phase HPLC column (4.6×250 mm, 7 μm particlesize) and compared to standards. The percentage of free drug compound inthe conjugate was determined as <0.5% of conjugated drug compound. SeeFIG. 22.

Preparation of Humanized Ab-SPDB-2a Conjugate

Humanized Ab at 8 mg/mL was derivatized with 4-6 molar equivalents ofSPDB hetrobifunctional linker for 1.5 h at 25′C in 95% PBS, PH 7.4,containing 5% DMA (v/v), and then purified over a G25 desalting columninto citrate buffer (35 mM citrate buffer, pH 5.5, containing 2 mM EDTA,150 mM NaCl) to remove unreacted linker. The LAR (Linker Antibody Ratio)were measured using UV absorbance at 280 and 343 nm without and with 50mM dithiothreitol addition (to measure total antibody anddithiothreitol-released SPy) and were determined to be 2.7-4.1 LAR. TheSPDB-modified antibody at 2 mg/mL was reacted with 2 molar equivalentsof compound 2a (HCl salt) per linked SPDB for 20 h at ambienttemperature in 85% citrate buffer, 15% DMA (v/v) and then purified overa G25 desalting column into PBS, pH 7.4 to remove unconjugated drugcompound. The DAR of the final humanized Ab-SPDB-2a conjugate wasmeasured by UV spectrophotometry at 280 and 350 nm and calculated to be˜1.7-2.1 DAR. The percentage of monomer and linked drug compound on themonomer in the conjugate was determined by HPLC using an SEC (sizeexclusion chromatography) column. See FIG. 23.

Example 7 In Vitro Potency of Free Drugs and Conjugates

General Procedure Used: Samples of unconjugated free drug compounds ordrug conjugates were added to 96-well flat bottomed tissue cultureplates and titrated using serial dilutions to cover the desired molarrange. Antigen positive (Ag⁺) or Antigen negative (Ag⁻) cells were addedto the wells in specific cell densities in such a way that there weretriplicate samples for each drug concentration for each correspondingcell line. The plates were then incubated at 37° C. in an atmosphere of5% CO₂ for 4-5 days depending on the cell line. COLO 205 (1,000cells/well), Namalwa (3,000 cells/well), HEL 92.1.7 (3,000 cells/well)—4days; RH30(1,000 cells/well), HL60/QC (5,000 cells/well), Ramos (10,000cells/well), KB (2,000 cells/well), BJAB (2,000 cells/well), NB4 (3,000cells/well)—5 days, RPMI 8226 (8,000 cells/well)—6 days.

At the end of the incubation period cytotoxic potencies were thenassessed using a WST-8 based cell viability assay and surviving cellswere measured by developing with WST-8 (2-7 hours). The absorbance ineach well was measured and the surviving fraction of cells at eachconcentration was plotted to reveal the cytotoxicity and/or antigenspecificity (of the conjugates).

Using the general procedure described above, the cytotoxicity of theunconjugated free drug compounds was measured against seven cell lines:KB, a HeLa cell contaminant, HL60/QC, an acute myeloid leukemia cellline, Namalwa, a Burkitt lymphoma cell line, NB4, an acute promyelocyticleukemia cell line, HEL92.1.7, an erythroleukemia cell line, RPMI8226, amultiple myeloma cell line and BJAB, a B-cell leukemia cell line. Theresults, shown in FIG. 24 and Table 10 demonstrate the high potency ofthese compounds across a wide range of cell types. The potency andspecificity of the antibody-drug conjugates were measured againstantigen-expressing cells, with and without the additions of an excessamount of blocking unconjugated antibody to show specificity of thekilling effect. The MY9-6-drug conjugate was extremely potent towardsthree different antigen-expressing cells: HL60/ATCC, HL60/QC and NB-4,despite the very low antigen expression in NB4 cells. The specificpotency could be blocked by addition of excess unconjugated antibody,demonstrating that the cell killing effect is antigen-specific.Similarly, the huFOLR1-drug conjugate was effective in killingantigen-expressing KB cells in a specific manner. Results areillustrated in FIGS. 25 and 26.

TABLE 10 Potency of free drugs against various cell lines. The IC₅₀values listed in the table are in the unit of nM. Namalwa KB HL60/QC NB4HEL92.1.7 RPMI8226 BJAB  1c 0.056 0.16 0.023  1d 0.069 0.18 0.032  1e2.4 >3.0 0.67 27d 0.23 0.05 0.039 0.14 0.07 0.04 27e 0.39 0.09 0.13 0.20.24 0.12 27f 4.4 1.7 1.1 1.8 >3.0 1 29a 0.002 0.004 0.001 0.0023 0.00310.011 0.001 29b 0.003 0.007 0.006 0.007 0.007 0.005 0.003 29c 0.0130.057 0.03 0.023 0.027 0.16 0.015

Similar results have also been obtained using different cell lines anddifferent conjugates of the invention, including: huMY9-6-SPDB-1fagainst HL60/QC (Ag⁺) cells, HL60/ATCC (Ag⁺) cells, and NB-4 (Ag⁺) cells(FIG. 25); huFOLR1-SPDB-1f against KB (Ag⁺) cells (FIG. 26);huMY9-6-SPDB-1f against antigen positive HL60/QC cells, HL60/ATCC cells,NB-4 cells, and HEL 92.1.7 cells (FIG. 29); huMy9-6-SPDB-1f,huMy9-6-sulfoSPDB-1f, and huMy9-6-BMPS-1f against HL60/QC (Ag⁺) cells(FIG. 34); chB38.1-SPDB-1f and chB38.1-sulfoSPDB-1f against COLO205(Ag⁺) cells (FIG. 35); huMy9-6-SPDB-1f, huMy9-6-sulfoSPDB-1f, andhuMy9-6-BMPS-1f against OCI-AML3 (Ag⁺) cells (FIG. 44). Also see FIG. 49for the potency of the various conjugates against various cell lines,expressed as IC₅₀ values (nM). Note that in FIGS. 25, 29, 34, 35, and44, conjugates were prepared in the presence of sodium bisulfite.

To compare in vitro potency measurements for the subject conjugatesprepared with and without imine reactive reagent, such as sodiumbisulfite, huMy9-6-BMPS-1f, huMy9-6-sulfo-SPDB-1f, and huMy9-6-Drug 2were prepared with and without sodium bisulfite using the in situsulfonation method (wherein the respective compounds of the inventionwas first mixed with sodium bisulfite and a bifunctional crosslinkerbearing a reactive group, then the reaction mixture, without furtherpurification, was reacted with the huMy9-6 monoclonal antibody as thecell-binding agent). IC₅₀s for the conjugates on HL60-QC cells are shownbelow. The data indicates that the inclusion of imine reactive group(such as sodium bisulfite) in the conjugate preparation step does notnegatively impact the in vitro potency of the subject conjugates.

NaHSO₃ IC₅₀ IC₅₀ (pM) huMy9-6 Conjugate treatment (pM) blockinghuMy9-BMPS-1f − 2 130 + 1.5 55 huMy9-6-sulfo-SPDB-1f − 5.6 1200 + 7.1610 huMy9-6-Drug 2 − 16 >3000 + 6.8 >3000

It is apparent that pre-treatment of the drug compounds with sodiumbisulfite (5 molar equivalents, 22 h, 4° C., 90:10 DMA:pH 5.5 water)prior to conjugation with huMy9-6 had no significant effect on theantigen dependent or antigen independent (antigen blocking with 1 μMunconjugated huMy9-6) in vitro potency of the conjugates.

Example 8 Binding of Antibody-Drug Conjugate is Similar to that ofUnmodified Antibody

The binding of huMY9-6-drug conjugate was compared with that of theunmodified huMY9-6 antibody against antigen-expressing HL60/QC cellsusing flow cytometry. Briefly, the antigen-positive cells were incubatedwith conjugates or unmodified antibodies at 4° C., then with a secondaryantibody-FITC conjugate at 4° C., fixed with formaldehyde (1% in PBS)and analyzed by flow cytometry. No significant difference was observedbetween the binding of the conjugate versus that of the unmodifiedantibody. An example is shown in FIG. 27, where a huMY9-6-drug conjugatebound to antigen-positive cells with a high affinity similar to that ofthe unmodified antibody.

Example 9 In Vivo Efficacy of huMY9-6-SPDB-1f Conjugate in HL60/QC TumorBearing Nude Mice

In this study, the anti-tumor activity of huMY9-6-SPDB-1f wasinvestigated in female nude mice bearing HL60/QC tumors, a human acutemyeloid leukemia model. HL60/QC tumor cells, 2×10⁶ cells/mouse weresubcutaneously inoculated at a volume of 0.1 mL/mouse in the area overthe right shoulder of female athymic nude mice, 5 weeks of age. Eightdays after tumor cell inoculation mice were randomized into groups (n=6per group) by tumor volume. Treatment was initiated the day ofrandomization, and groups included a control group dosed with PBS (200μL/injection), or a single treatment at various doses (5 to 100 μg/kg)of huMY9-6-SPDB-1f (50 μg/kg If dose corresponded to 2.5 mg/kg antibodydose). All treatments were well tolerated with the mean body weightlosses comparable to loss seen in PBS control mice. Mean tumor volume vstime is shown (FIGS. 28 and 36) with the data demonstrating adose-dependent anti-tumor activity of the huMY9-6-SPDB-1f conjugate. Theminimum effective dose was estimated to be 20 μg/kg, which is about35-fold lower that the maximum tolerated dose.

Example 10

The tolerability of huFOLR-1 conjugates was investigated in female CD-1mice. Animals were observed for seven days prior to study initiation andfound to be free of disease or illness. The mice were administered asingle i.v. injection of the conjugate and the animals were monitoreddaily for body weight loss, morbidity or mortality. Table 9 shows thatfor huFOLR1-drug1 the conjugate was tolerated at only the lowest dosetested of 50 μg/kg. In contrast, both mono-imine conjugates huFOLR1-Drug2 and huFOLR1-SPDB-1f were found to be better tolerated with a maximumtolerated dose of <198 μg/kg and >560 μg/kg respectively.

TABLE 9 Tolerability comparison data for (A) huFOLR1-drug 1, (B)huFOLR1-Drug 2, and (C) huFOLR1-SPDB-1f conjugates.

A) Dose (μg/kg) % Survival  50 100 100  0 200  0 300  0 400  0

B) Dose (μg/kg) % Survival  66 100 132 100 198  50 264  25

C) Dose (μg/kg) % Survival 120 100 160 100 200 100 320 100 560 100

Example 11

Compound 10:

To a stirred solution of if (18 mg, 0.019 mmol) andN-(β-maleimidopropyloxy)succinimide (BMPS) ester (9.2 mg, 0.034 mmol) inanhydrous dichloromethane (0.3 mL) was added anhydrousdiisopropylethylamine (5 μL, 0.029 mmol). The mixture was stirred atroom temperature for 27 hours, quenched with saturated ammonium chlorideand diluted with dichloromethane. The organic layer was washed withbrine, dried over anhydrous sodium sulfate, filtered and concentratedunder reduced pressure. The residue was purified by preparative reversephase HPLC (C18 column, CH₃CN/H₂O). The fractions containing productwere combined, extracted with dichloromethane and evaporated to givecompound 10 as a white solid (7.6 mg, y=33%). MS (m/z): found 1208.3(M+H)⁺. See FIG. 13.

Example 12

Compound 12:

To a stirred solution of if (16.5 mg, 0.018 mmol) and sulfo-SPDB (14.2mg, 0.036 mmol) in anhydrous dichloromethane (0.3 mL) was addedanhydrous diisopropylethylamine (9 μL, 0.054 mmol). The mixture wasstirred at room temperature overnight and concentrated under reducedpressure. The residue was purified by preparative reverse phase HPLC(C18 column, CH₃CN/H₂O). The fractions containing product were combined,extracted with dichloromethane and evaporated to give 6.6 mg of compound12 as yellowish foam. The aqueous layer was lyophilized to give another0.5 mg of compound 12 as white solid. MS (m/z): found 1235.0 (M−H)⁻. SeeFIG. 15.

Example 13 Preparation of Humanized Antibody-sulfoSPDB-1f Conjugate

A reaction containing 2.5 mg/mL huMy9-6 antibody and 10 molarequivalents of 10 (pretreated with 5-fold excess of sodium bisulfite in90:10 DMA:water) in 50 mM HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) pH 8.5 buffer and 15% v/v DMA(N,N-dimethylacetamide) cosolvent was allowed to conjugate for 6 hoursat 25° C. Post-reaction, the conjugate was purified and buffer exchangedinto 250 mM Glycine, 10 mM Histidine, 1% sucrose, 0.01% Tween, 50 μMsodium bisulfite formulation buffer, using NAP desalting columns(Illustra Sephadex G-25 DNA Grade, GE Healthcare). Dialysis wasperformed in the same buffer for 4 hours at room temperature utilizingSlide-a-Lyzer dialysis cassettes (ThermoScientific 20,000 MWCO). Thepurified conjugate was found to have a DAR of 2.4 (by UV-Vis using molarextinction coefficients ε_(330 nm)=15,484 cm⁻¹M⁻¹ and ε_(280 nm)=30, 115cm⁻¹M⁻¹ for 1f, and ε_(280 nm)=146,000 cm⁻¹M⁻¹ for My9-6 antibody),96.7% monomer (by size exclusion chromatography), <1% unconjugated freedrug compound (by acetone extraction/reverse-phase HPLC) and a finalprotein concentration of 1.4 mg/mL.

In vitro potency of antibody-sulfoSPDB-1f conjugates were measuredaccording to general procedure described in Example 7 and the data areshown in FIGS. 34 and 35. The antibody-sulfoSPDB-1f conjugates havecomparable or higher potency than the antibody-SPDB-1f conjugates.

Use of covalent imine reactants, such as sodium bisulfite, improvesAb-compound conjugate specifications (e.g., % monomer and drug load). Inone experiment, adduct formation was carried out with 5 molarequivalents of imine reactant over NHS-BMPS-1f in 90% DMSO/10% PBS pH7.4 for 4 h at 25° C. The reaction mixture was then added to huMy9-6antibody (4 molar equivalents IGN, 2 mg/ml, 10% v/v DMSO, 50 mM HEPESbuffer, pH 8.5, 5 h, 25° C.). Conjugates made using sodium hydrosulfite,sodium bisulfite, or sodium metabisulfite had similar IGN/Ab ratios and% monomer, while conjugates made with no additive treatment led to verylow drug incorporation. See table below.

IGN/Ab % monomer % 1f on Reactant (UV) (SEC) monomer Sodium 2.6 88 82Hydrosulfite Sodium Bisulfite 2.6 88 83 Sodium 2.7 88 82 MetabisulfiteNo additive 0.1 98 94

Example 14 Preparation of Humanized Antibody-BMPS-1f Conjugate

A reaction containing 2.0 mg/mL huMy9-6 antibody and 5 molar equivalentsof 12 (pretreated with 5-fold excess of sodium bisulfite in 90:10DMA:water) in 50 mM HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) pH 8.5 buffer and 15% v/v DMA(N,N-dimethylacetamide) cosolvent was allowed to react for 6 hours at25° C. Post-reaction, the conjugate was purified and buffer exchangedinto 250 mM Glycine, 10 mM Histidine, 1% sucrose, 0.01% Tween, 50 μMsodium bisulfite formulation buffer, using NAP desalting columns(Illustra Sephadex G-25 DNA Grade, GE Healthcare). Dialysis wasperformed in the same buffer for 4 hours at room temperature utilizingSlide-a-Lyzer dialysis cassettes (ThermoScientific 20,000 MWCO). Thepurified conjugate was found to have a DAR of 2.8 (by UV-Vis using molarextinction 146,000 cm⁻¹M⁻¹ for My9-6 antibody), 91.7% monomer (by sizeexclusion chromatography), <1% unconjugated free drug compound (byacetone extraction/reverse-phase HPLC) and a final protein concentrationof 1.2 mg/mL.

In vitro potency of antibody-BMPS-1f conjugates were measured accordingto general procedure described in Example 7 and the data are shown inFIGS. 34 and 35. The antibody-BMPS-1f conjugates have comparable potencyto the antibody-SPDB-1f conjugates.

Example 15 In Vivo Efficacy of Hu FOLR1-SPDB-1f Conjugate in KB TumorBearing Nude Mice

In this study, the anti-tumor activity of hu FOLR1-SPDB-1f wasinvestigated in female nude mice bearing KB tumors, a human cervicalcarcinoma model. KB, 1×10⁷ cells/mouse were subcutaneously inoculated ata volume of 0.1 mL/mouse in the area over the right shoulder of femaleathymic nude mice, 6 weeks of age. Six days after tumor cell inoculationmice were randomized into groups (n=6 per group) by tumor volume.Treatment was initiated the day after randomization, and groups includeda control group dosed with PBS (200 μL/injection), or a single treatmentat various doses (20 to 200 μg/kg) of hu FOLR1-SPDB-1f (50 μg/kg linkeddrug dose corresponded to 2.8 mg/kg antibody dose). All treatments werewell tolerated with no body weight loss seen in any of the test groups.Mean tumor volume vs time is shown (FIG. 37) with the data demonstratinga dose-dependent anti-tumor activity of the hu FOLR1-SPDB-1f conjugate.The minimum effective dose was estimated to be <50 μg/kg, which is about14-fold lower than the maximum tolerated dose.

Similar in vivo results have also been obtained using other conjugatesof the invention against various other cancer models, includinghuMy9-6-sulfo-SPDB-1f in MOLM-13 tumor bearing mice (FIG. 50);huMy9-6-sulfo-SPDB-1f in NB4 tumor bearing mice (FIG. 51);huMy9-6-BMPS-1f in HL60/QC tumor bearing mice (FIG. 52); huMy9-6-BMPS-1fin MOLM-13 tumor bearing mice (FIG. 53); huMy9-6-Drug 2 in HL60/QC tumorbearing mice (FIG. 56); and huMy9-6-Drug 2 in MOLM-13 tumor bearing mice(FIG. 57). Note that in FIGS. 53, 54, 56, and 57, conjugates wereprepared in the presence of sodium bisulfite.

To compare in vivo efficacy of the subject conjugates prepared with orwithout an imine reactive group, huMy9-6-Drug 2 were formulated with orwithout 50 μM sodium bisulfite, and the conjugates were used to treatmice bearing HL60-QC tumor xenografts. The data below shows thatconjugate formulated with or without 50 μM sodium bisulfite showedcomparable T/C % at ˜20 μg/kg drug dose, indicating that the inclusionof sodium bisulfite in the conjugate preparation step does notnegatively impact the in vivo potency of the subject conjugate.

Minimum effective dose NaHSO₃ (μg/kg drug) T/C % − 18 20 + 19 16

Example 16

Compound 27b:

(5-((2-mercapto-2-methylpropylthio)methyl)-1,3-phenylene)dimethanol:(5-(mercaptomethyl)-1,3-phenylene)dimethanol (0.163 g, 0.885 mmol) wasdissolved in methanol (3 mL) in a small vial and a stir bar was added.To this solution was added triethylamine (0.016 mL, 0.118 mmol) followedby 2,2-dimethylthiirane (0.058 mL, 0.590 mmol) and the resulting mixturewas capped and stirred overnight (16 hrs) at room temperature. Thereaction was then concentrated, redissolved in dichloromethane, loadedonto a silica ptlc plate (1000 micron) and the plate was developed using10% methanol in dichloromethane. The band corresponding to the productwas scraped, filtered with neat ethyl acetate, and concentrated to give(5-((2-mercapto-2-methylpropylthio)methyl)-1,3-phenylene)dimethanol(0.095 g, 0.349 mmol, 59.1% yield). ¹H NMR (400 Hz, CDCl₃): δ 7.26 (s,3H), 4.69 (s, 4H), 3.82 (s, 2H), 2.74 (s, 2H), 2.17 (s, 1H), 2.12 (br s,2H), 1.43 (s, 6H); ¹³C NMR (400 Hz, CDCl₃): δ 141.6, 138.9, 126.7,124.3, 65.0, 49.0, 45.4, 38.4, 31.5; MS (m/z), expected: 272.4, found295.0 (M+Na). See FIG. 30.

Compound 27c:

(5-((2-methyl-2-(methyldisulfanyl)propylthio)methyl)-1,3-phenylene)dimethanol:(5-((2-mercapto-2-methylpropylthio)methyl)-1,3-phenylene)dimethanol(0.120 g, 0.440 mmol) was dissolved in ethanol (5 mL) and 1.0 Mpotassium phosphate buffer (pH 7) (5.00 mL) and cooled in an ice bath (appt formed but it was ignored). S-methyl methanesulfonothioate (0.083mL, 0.881 mmol) was added and the mixture stirred overnight with gradual(over 30 minutes) warming to room temperature. The reaction was dilutedwith dichloromethane and the organic layer was removed, washed withwater, dried over anhydrous sodium sulfate, and concentrated in vacuo.The residue was dissolved in dichloromethane and loaded onto a 500micron ptlc plate and developed with 66% ethyl acetate in hexane. Theband corresponding to the product was scraped, filtered using ethylacetate, and concentrated to give(5-((2-methyl-2-(methyldisulfanyl)propylthio)methyl)-1,3-phenylene)dimethanol(0.091 g, 0.286 mmol, 64.9% yield). ¹H NMR (400 Hz, CDCl₃): δ 7.27 (s,3H), 4.71 (s, 4H), 3.78 (s, 2H), 2.77 (s, 2H), 2.41 (s, 3H), 1.94 (br s,2H), 1.38 (s, 6H); ¹³C NMR (400 Hz, CDCl₃): δ 141.6, 139.0, 126.7,124.2, 65.0, 51.8, 44.0, 38.2, 26.7, 25.3; MS (m/z), expected: 341.5,found 341.1 (M+Na). See FIG. 30.

Compound 27d:

(5-((2-methyl-2-(methyldisulfanyl)propylthio)methyl)-1,3-phenylene)dimethanol(80 mg, 0.251 mmol) in anhydrous dichloromethane (1.75 mL) was cooled to−5° C. in a brine/ice bath. Triethylamine (105 μL, 0.753 mmol) was addedfollowed by the addition of methanesulfonyl chloride (50.7 μL, 0.653mmol) at −5° C. The reaction was stirred at −5° C. for one hour afterwhich it was diluted with cold ethyl acetate and ice was added. Themixture was transferred to a separatory funnel and extracted with coldethyl acetate. The organic extracts were washed with ice water and thendried with anhydrous magnesium and sodium sulfate, filtered andconcentrated under reduced pressure. The resulting(5-((2-methyl-2-(methyldisulfanyl)propylthio)methyl)-1,3-phenylene)bis(methylene)dimethanesulfonatewas used without further purification.

IBD monomer (177 mg, 0.602 mmol) in anhydrous N,N-dimethylformamide(1.75 mL) was added to(5-((2-methyl-2-(methyldisulfanyl)propylthio)methyl)-1,3-phenylene)bis(methylene)dimethanesulfonate(119 mg, 0.251 mmol) at ambient temperature. Potassium carbonate (173mg, 1.253 mmol) was added and the reaction was allowed to stir atambient temperature for 20 hours. The reaction mixture was quenched withwater and extracted with dichloromethane. The extracts were washed withbrine and then dried with anhydrous sodium sulfate, filtered andconcentrated on high vacuum. The crude product was purified by flashsilica gel chromatography (neat DCM 2% MeOH/DCM). Fractions containingproduct were combined, concentrated and purified by semi-prep RP-HPLC(C18, A=DI water B=ACN, 20 mL/min). Fractions containing desired productwere combined, extracted with dichloromethane, dried with anhydrousmagnesium sulfate, filtered and concentrated to yield the desiredproduct (46 mg, 21%). ¹H NMR (400 Hz, CDCl₃): δ 8.19 (d, J=8.0 Hz, 2H),7.77 (m, d, J=4.4 Hz, 2H), 7.50 (s, 2H), 7.34 (s, 1H), 7.31 (s, 2H),7.19 (m, 4H), 7.03 (t, J=7.2, 7.6 Hz, 2H), 6.77 (s, 2H), 5.14 (m, 4H),4.40 (m, 2H), 3.91 (s, 6H), 3.70 (m, 2H), 3.63 (m, 2H), 3.41 (m, 2H),2.65 (s, 2H), 2.29 (s, 3H), 1.26 (s, 6H). MS (m/z), Calcd. 893.2(M+Na)⁺. found 893.2 (M+Na)⁺, 911.2 (M+H₂O+Na)⁺, 929.2 (M+2H₂O+Na)⁺,945.1 (M+2H₂O+K)⁺. See FIG. 30.

Compound 27e and 27f:

To a cooled solution (0° C.) of 27d (50 mg, 0.057 mmol) in anhydrousdichloromethane (225 μL) and ethanol (450 μL) was added sodiumborohydride (0.651 mg, 0.017 mmol). The reaction was stirred for fiveminutes at 0° C. and then at ambient temperature for 2.5 hrs. Thereaction mixture was cooled to 0° C., quenched with saturated ammoniumchloride, and extracted with dichloromethane. The organic extracts werewashed with brine, dried over anhydrous sodium sulfate, filtered throughCelite and concentrated. The crude material was purified by semi-prepRP-HPLC (C18, A=DI water B=ACN, 20 mL/min). Fractions containing desiredproduct were combined, extracted with dichloromethane, dried withanhydrous magnesium sulfate, filtered and concentrated to yield the monoreduced amine 27e (11 mg, 22%) MS (m/z), Calcd. 895.3 (M+Na)⁺ found895.2 (M+Na)⁺, 913.2 (M+H₂O+Na)⁺, 929.2 (M+H₂O+K)⁺ and the di-reducedamine 27f (5 mg, 10%) MS (m/z), Calcd. 897.3 (M+Na)⁺. found 897.3(M+Na)⁺. See FIG. 30.

Compound 27g:

To a stirred solution of 27e (10 mg, 0.011 mmol) in methanol (733 μL)and acetonitrile (880 μL) was added tris(2-Carboxyethyl) phosphinehydrochloride (9.85 mg, 0.034 mmol) in buffer 6.5 (147 μL). The mixturestirred at 3 hours at ambient temperature. The reaction was diluted withdichloromethane. Water was added and the layers were separated. Theorganic layer was washed with brine, dried with anhydrous sodiumsulfate, filtered and concentrated under reduced pressure to givecompound 27g (9 mg, 95%). MS (m/z), Calcd. 849.3 (M+Na)⁺. found 849.2(M+Na)⁺, 867.2 (M+K)⁺. See FIG. 30.

Compound 27h:

To a stirred solution of 27g (9 mg, 10.88 μmol) and2,5-dioxopyrrolidin-1-yl 4-(pyridin-2-yldisulfanyl)butanoate (9.3 mg,0.023 mmol) in anhydrous dichloromethane (0.4 mL) was added anhydrousdiisopropylethylamine (9 μl, 0.054 mmol) and the reaction was stirred atroom temperature overnight. The mixture was quenched with saturatedammonium chloride solution and extracted with dichloromethane. Theextracts were washed with brine, dried with anhydrous sodium sulfate andconcentrated under reduced pressure. The residue was purified bypreparative reverse phase HPLC (C18 column, CH₃CN/H₂O). The fractionscontaining product were combined, extracted with dichloromethane andevaporated to give compound 27h (5 mg, 44%). MS (m/z), Calcd. 1064.3(M+Na)⁺. found 1064.1 (M+Na)⁺, 1082.1 (M+H₂O+Na)⁺, 1098.1 (M+H₂O+K)⁺.See FIG. 30.

Example 17

Compound 28b:

(5-(methyl(2-methyl-2-(methyldisulfanyl)propyl)amino)-1,3-phenylene)dimethanol(52 mg, 0.172 mmol) was dissolved in anhydrous dichloromethane (1.7 mL)and cooled to −5 in an acetone/ice bath. First, triethylamine (0.120 mL,0.862 mmol) was added followed by methanesulfonyl chloride (0.040 mL,0.517 mmol). The mixture was stirred in the bath for 1 hour. Thereaction was then diluted with cold ethyl acetate and washed with coldwater three times and then dried over anhydrous magnesium sulfate. Thedimesylate was filtered, concentrated in vacuo, and placed under highvacuum until completely dry. The product was used as is directly in thenext step.

IBD monomer (115 mg, 0.39 mmol) in anhydrous N,N-dimethylformamide (1.5mL) was added to(5-(methyl(2-methyl-2-(methyldisulfanyl)propyl)amino)-1,3-phenylene)bis(methylene)dimethanesulfonate(72 mg, 0.156 mmol) at ambient temperature. Potassium carbonate (108 mg,0.780 mmol) was added and the reaction was allowed to stir at ambienttemperature for 20 hours. Water (10 mL) was added directly to themixture with stirring resulting in the formation of a white precipitate.The mixture was filtered and the solids were washed with additionalportions of water. The solid was then dissolved in dichloromethane,extracted with water, the organic layer was then dried over anhydroussodium sulfate, filtered, and concentrated in vacuo to give compound 28b(104 mg, 78%) which was used in the next step without further treatment.MS (m/z), found 912.1 (M+2H₂O+Na). See FIG. 31.

Compound 28c and 28d:

Compound 28b (55 mg, 0.064 mmol) was dissolved in an anhydrous mixtureof dichloromethane (0.4 mL) and ethanol (0.8 mL) and cooled to 0° C. inan ice bath. A sodium borohydride (0.731 mg, 0.019 mmol) solutiondissolved in ethanol (100 μl) was then added and the mixture was stirredfor 5 minutes and the ice bath was removed. The reaction was allowed tostir for 2 hours, quenched at low temperature by adding saturatedammonium chloride and dichloromethane, separated and the organic layerwas washed with brine, dried over anhydrous sodium sulfate, filtered,and concentrated in vacuo. The residue was purified by semi-prep RP-HPLC(C18, A=DI water B=ACN, 20 mL/min). The fractions containing the desiredproducts were extracted with dichloromethane and concentrated to givethe mono-imine 28c (19 mg, 32%) MS (m/z), expected: 855.1, found: 896.2(M+H₂O+Na) and the di-reduced amine 28d (22 mg, 38%) MS (m/z), expected:857.1, found: 880.2 (M+Na)⁺. See FIG. 31.

Example 18

Compound 29b and 29c:

Compound 29a (60 mg, 0.043 mmol) was dissolved in an anhydrous mixtureof dichloromethane (0.25 mL) and ethanol (0.5 mL) and cooled to 0° C. inan ice bath. A sodium borohydride (0.493 mg, 0.013 mmol) solutiondissolved in ethanol (50 μL) was then added and the mixture was stirredfor 5 minutes and the ice bath was removed. The reaction was allowed tostir for 3 hours, quenched at low temperature by adding saturatedammonium chloride and dichloromethane, separated and the organic layerwas washed with brine, dried over anhydrous sodium sulfate, filtered,and concentrated in vacuo. The residue was purified by semi-prep RP-HPLC(C18, A=DI water B=ACN, 20 mL/min). The fractions containing the desiredproducts were extracted with dichloromethane and concentrated to givethe mono-imine 29b (20 mg, 33%) MS (m/z), expected: 715.7, found: 715.2(M+Na)⁺, 733.2 (M+H₂O+Na)⁺, 749.2 (M+H₂O+K)⁺ and the di-reduced amine29c (12 mg, 20%) MS (m/z), expected: 694.7, found: 717.2 (M+Na)⁺. SeeFIG. 32.

Example 19

Compound 30a:

(5-((2-(2-(2-methoxyethoxy)ethoxy)ethyl)(2-methyl-2-(methyldisulfanyl)propyl)amino)-1,3-phenylene)bis(methylene)dimethanesulfonate(0.566 g, 0.960 mmol) was dissolved in acetone (30 mL) and a solution ofsodium iodide (0.544 g, 3.63 mmol) dissolved in acetone (2 mL) was addedwith vigorous stirring. The reaction was monitored by tlc (50% ethylacetate in hexane) and after 2 hours the reaction was filtered,concentrated in vacuo and dichloromethane was added to the residue. Thesolid salt left behind was filtered, the filtrate was concentrated andthe resulting residue was purified on silica gel using a 3:5:2 mixtureof ethyl acetate:hexane:dichloromethane to give3,5-bis(iodomethyl)-N-(2-(2-(2-methoxyethoxy)ethoxy)ethyl)-N-(2-methyl-2-(methyldisulfanyl)propyl)aniline(0.505 g, 0.773 mmol, 74.5% yield) as a yellow oil. ¹H NMR (400 Hz,CDCl₃): δ 6.75 (s, 2H), 6.73 (s, 1H), 4.38 (s, 4H), 3.63 (m, 14H), 3.40(s, 3H), 2.50 (s, 3H), 1.38 (s, 6H); ¹³C NMR (400 Hz, CDCl₃): δ 148.7,140.3, 117.3, 113.4, 71.9, 70.7, 70.6, 67.2, 59.8, 59.1, 53.5, 53.4,51.8, 26.5, 25.6, 6.11; MS (m/z), Calcd 676.0 (M+Na)⁺. found 675.8(M+Na)⁺. See FIG. 33.

Compound 30b:

IBD Monomer (0.060 g, 0.204 mmol) was dissolved in acetone (4 ml) in asmall vial, a stir bar was added, followed by3,5-bis(iodomethyl)-N-(2-(2-(2-methoxyethoxy)ethoxy)ethyl)-N-(2-methyl-2-(methyldisulfanyl)propyl)aniline(0.167 g, 0.255 mmol) and potassium carbonate (0.070 g, 0.510 mmol). Thevial was capped and stirred at room temperature overnight. The solidswere filtered off and the filtrate was concentrated. The residue wasdissolved in dichloromethane, extracted with water, and the organiclayer was dried over anhydrous sodium sulfate, filtered, andconcentrated in vacuo to give 108 mg of crude material. The crudematerial was purified on silica gel using 30% ethyl acetate to removethe di-iodo starting material followed by 10% methanol indichloromethane to give the desired product 30b (21 mg, 0.026 mmol,13%). MS (m/z), expected: 819.1, found: 858.0 (M+K)⁺, 890.0(M+CH₃OH+K)⁺. See FIG. 33.

Compound 1d:

The reduced monomer 3b (4.16 mg, 0.014 mmol) was dissolved in acetone (2ml) in a small vial, a stir bar was added, followed by 30b (10 mg, 0.012mmol) and potassium carbonate (4.21 mg, 0.030 mmol). The vial was cappedand stirred at room temperature overnight. The reaction was concentratedto remove the acetone and then redissolved in dichloromethane, extractedwith water, dried over anhydrous sodium sulfate, filtered, andconcenrated in vacuo. The residue was purified by reverse phase C18 HPLCto get 1d (2.1 mg, 2.125 μmol, 17.42% yield). MS (m/z): found 1010.4(M+Na)⁺, 1028.4 (M+H₂O+Na)⁺. See FIG. 33.

Example 20 Synthesis of Compound 1

Compound 1:

To a stirred suspension of if (226 mg, 0.24 mmol) in IPA (20 mL) anddeionized water (10 mL) was added sodium bisulphite (50 mg, 0.48 mmol).The mixture was stirred vigorously at rt for 2 hours. It was frozen withdry ice/acetone and lyophilized. The obtained white fluffy solid wasdissolved in CH₃CN/H₂O and purified by reverse phase HPLC (C18 column,CH₃CN/H₂O). The fractions containing the desired product were combinedand frozen with dry ice/acetone and lyophilized to give the desiredcompound 1 as white fluffy solid (179.6 mg, 5=71.6%). MS (m/z): found1022.0 (M−H)⁻. See FIG. 38.

Example 21 Synthesis of Compound 9c

Compound 9c:

To a stirred solution of 1c (60 mg, 0.061 mmol) in CH₃CN (3 mL) wasadded freshly prepared TCEP solution (49 mg, 0.17 mmol of TCEP HCl saltwas neutralized with saturated sodium bicarbonate to pH ˜6.5 thendiluted with 0.5 mL of pH 6.5 phosphate buffer) at room temperature.MeOH (2.5 mL) was added and the mixture was stirred at room temperaturefor 3 hours. The reaction mixture was diluted with dichloromethane anddeionized water, separated and the organic layer was washed with brine,dried over anhydrous Na₂SO₄ and filtered. The filtrate was stripped andhigh vacuumed to give 60 mg of 1 h as light yellowish foam. MS (m/z):found 940.1 (M+H)⁺. It was dissolved in methanol (1.0 mL) and CH₃CN (1.4mL) followed by addition of iodoacetic acid (24 mg, 0.13 mmol),deionized water (0.1 mL) and potassium carbonate (27 mg, 0.19 mmol). Themixture was stirred at rt overnight (monitored by LCMS). It was quenchedwith saturated ammonium chloride to make the solution acidic thendiluted with dichloromethane, separated and washed with brine, driedover anhydrous Na₂SO₄, filtered and stripped to give compound 9c (57.8mg, y=91%) which was directly used for next step without purification.MS (m/z): found 998.1 (M+H)⁺. See FIG. 12A.

Compound 9a:

To a stirred solution of compound 9c (57.8 mg, 0.058 mmol) in anhydrousdichloromethane (0.2 mL) and absolute ethanol (0.6 mL) was added NaBH₄(2.5 mg, 0.066 mmol) at 0° C. The ice bath was removed and the mixturewas stirred at room temperature for 3 hours and then quenched withsaturated ammonium chloride, diluted with dichloromethane, separated andthe organic layer was washed with brine, dried over anhydrous Na₂SO₄ andfiltered through celite and stripped. The residue was purified byreverse phase HPLC (C18 column, CH₃CN/H₂O). The product fractions wereextracted with dichloromethane and stripped to give compound 9a (13.0mg, y=22%). MS (m/z): found 1000.0 (M+H)⁺, 1015.9 (M+H₂O−H)⁻. See FIG.12A.

Compound 9a:

To a solution of the free thiol if (45 mg, 0.048 mmol) and iodoaceticacid (18 mg, 0.096 mmol) in methanol (1.0 mL) and CH₃CN (1.4 mL) wasadded deionized water (0.1 mL) and potassium carbonate (20 mg, 0.14mmol). The mixture was stirred at rt overnight (monitored by LCMS). Itwas quenched with saturated ammonium chloride to make the solutionacidic then diluted with dichloromethane, separated and washed withbrine, dried over anhydrous Na₂SO₄, filtered and stripped. The residuewas purified by preparative reverse phase HPLC (C18, CH₃CN/H₂O). Thepure product fractions (based on MS) were extracted withdichloromethane, stripped to give the desired acid 9a (18 mg, y=38%). MS(m/z): found 1000.1 (M+H)⁺. See FIG. 12B.

Example 22 Synthesis of Compound 1d

Compound 1d:

To a stirred solution of compound 1c (178 mg, 0.18 mmol) in anhydrousdichloromethane (1.2 mL) and absolute ethanol or anhydrous methanol (0.1mL) was added 5-ethyl-2-methylpyridine borane (PEMB, 0.017 mL, 0.11mmol) dropwise. The mixture was stirred at rt for 1 hour and quenchedwith 88% formic acid. It was basified with saturated NaHCO₃ and dilutedwith dichloromethane, separated and the organic layer was washed withbrine, dried over anhydrous Na₂SO₄ and filtered through celite andstripped. The residue was dissolved in CH₃CN/H₂O/88% HCOOH (5:1:0.05)and purified by reverse phase HPLC (C18, CH₃CN/H₂O). The fractions thatcontained pure product were extracted with dichloromethane and strippedto give compound 1d (56 mg, y=31%). MS (m/z): found 988.1 (M+H)⁺. SeeFIG. 39.

Compound 1d:

To a stirred solution of compound 1c (71 mg, 0.072 mmol) in anhydrous1,2-dichloroethane (0.8 mL) was added sodium triacetoxyborohydride (14mg, 0.65 mmol). The mixture was stirred at rt for 2 hours and quenchedwith saturated NaHCO₃ and diluted with dichloromethane, separated andthe organic layer was washed with brine, dried over anhydrous Na₂SO₄ andfiltered through celite and stripped. The residue was dissolved inCH₃CN/H₂O/88% HCOOH (5:1:0.05) and purified by reverse phase HPLC (C18,CH₃CN/H₂O). The fractions that contained pure product were extractedwith dichlormethane and stripped to give compound 1d (17 mg, y=24%). MS(m/z): found 988.1 (M+H)⁺. Unreacted starting material 1c was alsorecovered (24 mg, y=34%). See FIG. 40.

Example 23 Synthesis of Compound 31c

Compound 31a:

To a solution of compound 1f (57.8 mg, 0.061 mmol) and methyl4-bromobutyrate (22 mg, 0.12 mmol) in methanol (1.0 mL) and CH₃CN (1.0mL) was added deionized water (0.1 mL) and potassium carbonate (17 mg,0.12 mmol). The mixture was stirred at rt overnight then quenched withsaturated ammonium chloride and diluted with dichloromethane, separatedand washed with brine, dried over anhydrous Na₂SO₄, filtered andstripped. The residue was purified by preparative reverse phase HPLC(C18, CH₃CN/H₂O) to give the desired product 31a (14 mg, y=22%) asyellowish foam. MS (m/z): found 1042.1 (M+H)⁺. See FIG. 41.

Compound 31b:

To a solution of the methyl ester 31a (14 mg, 0.013 mmol) in anhydrous1,2-dichloroethane (1.5 mL) was added trimethyltin hydroxide (36 mg, 0.2mmol). The mixture was stirred overnight in a 80° C. oil bath untilstarting material was completely consumed. It was cooled to roomtemperature, diluted with dichloromethane, washed with brine/drops 5%HCl and brine, dried and filtered. The filtrate was stripped andpurified with silica gel chromatography (dichloromethane/MeOH) to giveacid 31b as yellowish solid (10.2 mg, y=74%). MS (m/z): found 1028.2(M+H)⁺, 1044.1 (M+H₂O−H)⁻. See FIG. 41.

Compound 31c:

To a solution of acid 31b (10.2 mg, 0.0099 mmol) in anhydrousdichloromethane (0.5 mL) was added N-hydroxysuccinimide (3.4 mg, 0.03mmol) and PL-DCC (26 mg, 0.04 mmol, 1.55 mmol/g). The mixture wasstirred at room temperature overnight and filtered to remove the resin.The resin was washed with dichloromethane then ethyl acetate. Thefiltrate was stripped and the residue was purified by reverse phase HPLC(C18, CH₃CN/H₂O). The fractions containing product were combined andlyophilized to give NHS ester 31c as white solid (3.6 mg, y=32%). MS(m/z): found 1125.1 (M+H)⁺. See FIG. 41.

Example 24 Synthesis of Compound 32c

Compound 32a:

To a stirred solution of the aniline 1a (339 mg, 1.1 mmol) in anhydroustetrahydrofuran (4.0 mL) was added Boc anhydride (272 mg, 1.2 mmol). Themixture was continued to be stirred at room temperature for three days.The reaction mixture was concentrated under reduced pressure and theresidue was purified by silica gel chromatography (CH₂Cl₂/MeOH) to givecompound 32a (405 mg, y=90%) as colorless oil. ¹H NMR (400 Hz, CDCl₃): δ7.00 (s, 2H), 6.97 (s, 1H), 4.38 (s, 4H), 4.12 (s, 2h), 3.64 (t, J=5.6Hz, 2H), 3.48-3.44 (m, 8H), 3.40-3.38 (m, 2H), 3.21 (s, 3H), 1.31 (s,9H); ¹³C NMR (400 Hz, CDCl₃): δ 154.65, 142.3, 142.1, 124.1, 122.7,80.2, 71.6, 70.3, 70.1, 69.9, 68.5, 63.9, 58.65, 49.4, 28.1. See FIG.42.

Compound 32b:

To a stirred solution of compound 32a (51 mg, 0.128 mmol) in anhydrousdichloromethane was added triethylamine (0.053 mL, 0.383 mmol) at−5˜−10° C. Methansulfonyl chloride (0.026 mL, 0.332 mmol) was then addedslowly in 15 minutes with a syringe. The mixture was stirred at −5-10°C. for 1 hours (TLC, DCM/MeOH 10:1). The reaction was quenched withice/water, diluted with cold AcOEt, separated and the organic layer waswashed with cold water, dried over anhydrous Na₂SO₄/MgSO₄, filtered andstripped. The residue was transferred into a small reaction flask withdichloromethane, stripped and high vacuumed. It was dissolved inanhydrous DMF (0.8 mL) followed by addition of IBD monomer (90 mg, 0.31mmol) and potassium (53 mg, 0.38 mmol). The mixture was stirred at rtovernight. It was diluted with dichloromethane, washed with brine, driedover anhydrous sodium sulfate, filtered and stripped. The residue waspurified by reverse phase HPLC (C18, CH₃CN/H₂O) to give compound 32b (56mg, 46%) as yellowish solid. %). ¹H NMR (400 Hz, CDCl₃): δ 8.29 (d,J=8.0 Hz, 2H), 7.87 (d, J=4.8 Hz, 2H), 7.60 (s, 2H), 7.38-7.36 (m, 3H),7.33-7.27 (m, 4H), 7.13 (t, J=7.6 Hz, 2H), 6.88 (s, 2H), 5.21 (dd,J₁=20.0 Hz, J₂=12.4 Hz, 4H), 4.49 (dt, J₁=11.2 Hz, J₂=4.0 Hz, 2H), 3.99(s, 6H), 3.83 (t, J=6.0 Hz, 2H), 3.76-3.48 (m, 14H), 3.35 (s, 3H), 1.43(s, 9H); MS (m/z): found 992.2 (M+H₂O+Na)⁺, 1010.2 (M+2H₂O+Na)⁺. SeeFIG. 42.

Compound 32c:

To a stirred solution of compound 32b (56 mg, 0.059 mmol) in anhydrousdichloromethane (0.3 mL) and absolute ethanol (0.9 mL) was added NaBH₄(2.7 mg, 0.07 mmol) at 0° C. The ice bath was removed and the mixturewas stirred at room temperature for 3 hours and then quenched withsaturated ammonium chloride, diluted with dichloromethane, separated andthe organic layer was washed with brine, dried over anhydrous Na₂SO₄ andfiltered through celite and stripped. The residue was purified byreverse phase HPLC (C18 column, CH₃CN/H₂O). Recovered starting material32b weighed 12 mg which was re-subjected to the reduction conditions andpurified by reverse phase HPLC. All the fractions that contained pureproduct were extracted with dichloromethane and stripped to givecompound 32c (20.7 mg, y=37%) as a light yellowish solid. MS (m/z):found 954.2 (M+H)⁺. See FIG. 42.

Example 25

The tolerability of huMy9-6 conjugates was investigated in female CD-1mice. Animals were observed for seven days prior to study initiation andfound to be free of disease or illness. The mice were administered asingle i.v. injection of the conjugate and the animals were monitoreddaily for body weight loss, morbidity or mortality. Table 10 shows thatthe huMy9-6-SPDB-1c di-imine disulfide containing conjugate wastolerated at a dose of less than 300 μg/kg. In contrast, the mono-iminedisulfide conjugates huMy9-6-SPDB-1f and huMy9-6-sulfo-SPDB-1f werefound to be better tolerated with a maximum tolerated dose of >729 μg/kgand <750 μg/kg respectively.

TABLE 10 Tolerability comparison data for (A) huMy9-6-SPDB-1c, (B)huMy9-6-SPDB- 1f, (C) huMy9-6-sulfo-SPDB-1f, and (D) huMy9-6-BMPS-1fconjugates.

A) Dose (μg/kg) % Survival 100 100 300  50 500  0 700  0

B) Dose (μg/kg) % Survival 405 100 567 100 729 100

C) Dose (μg/kg) % Survival 450 100 600 100 750  88 900  50

D) Dose (μg/kg) % Survival 100 100 200 100 284 100 324  83 405  50

Example 26

Compound 33b:

Compound 33a (20 g, 77 mmol) was added as a thick suspension inanhydrous dichloromethane (100 mL) and was cooled to 0° C. Acetic acid(191 mL) was added, resulting in a clear solution which stirred at 0° C.until cool. Nitric acid (26 mL, 581 mmol) was added slowly dropwisethrough an addition funnel. The ice bath was removed and the solutioncontinued to stir at room temperature. After 3 hours, the reaction wasdiluted with deionized water and extracted with dichloromethane. Theorganic layer was washed with brine, dried over anhydrous magnesiumsulfate and the filtrate concentrated in vacuo. The crude residue wasrecrystallized using ethyl acetate and hexanes. The solid was filteredand washed with hexanes to give compound 33b as a yellow fluffy solid(13.8 g, y=59%). ¹H NMR (400 Hz, CDCl₃): δ 7.48-7.43 (m, 6H), 7.25 (s,1H), 5.25 (s, 2H), 4.02 (s, 3H), MS (m/z): 326.1 (M+Na)⁺. See FIG. 45.

Example 27

3,5-bis(((tert-butyldimethylsilyl)oxy)methyl)-N-(2-(2-(2-methoxyethoxy)ethoxy)ethyl)aniline

A mixture of (5-amino-1,3-phenylene)dimethanol (11.78 g, 77 mmol),2-(2-(2-methoxyethoxy)ethoxy)ethyl 4-methylbenzenesulfonate (15.3 g,48.1 mmol), and potassium carbonate (13.28 g, 96 mmol) in DMF (96 ml)was refluxed for 20 hours. The reaction was cooled to ambienttemperature and diluted with dichloromethane. The mixture was filteredthrough celite and concentrated in vacuo. The resulting orange oil wasdissolved in dichloromethane (240 ml) and t-butyldimethylsilyl chloride(18.09 g, 120 mmol) and imidazole (9.80 g, 144 mmol) were added. Thereaction was stirred at ambient temperature for 20 hours upon which itwas diluted with dichloromethane and filtered through celite.Purification by silica gel chromatography (EtOAc/Hex) yielded3,5-bis(((tert-butyldimethylsilyl)oxy)methyl)-N-(2-(2-(2-methoxyethoxy)ethoxy)ethyl)aniline(13g, 52%). ¹H NMR (400 Hz, CDCl₃): δ6.52 (s, 1H), 6.40 (s, 2H), 4.56(s, 4H), 3.60 (t, 2H, J=5.2 Hz), 3.56 (m, 6H), 3.46 (m, 2H), 3.29 (s,3H), 3.20 (t, 2H, J=5.2 Hz), 0.84 (s, 18H), 0.00 (s, 12H). MS (m/z):found 550.1 (M+Na)⁺. See FIG. 46.

3,5-bis(((tert-butyldimethylsilyl)oxy)methyl)-N-(2-(2-(2-methoxyethoxy)ethoxy)ethyl)-N-(2-methyl-2-(methyldisulfanyl)propyl)aniline

To a solution of3,5-bis(((tert-butyldimethylsilyl)oxy)methyl)-N-(2-(2-(2-methoxyethoxy)ethoxy)ethyl)aniline(6.7 g, 12.69 mmol) in anhydrous 1,2-dichloroethane (50 ml) was added2-(methyldithio)isobutyraldehyde (2.74 ml, 19.04 mmol), sodiumtriacetoxyborohydride (2.8 g, 1 eq), zinc(II) chloride (0.865 g, 6.35mmol) and magnesium sulfate (2.292 g, 19.04 mmol). The mixture wasstirred for five hours at ambient temperature. Sodiumtriacetoxyborohydride (2.8 g, 1 eq) was added. The reaction continued tostir at ambient temperature for 20 hours. The mixture was filteredthrough celite rinsing with dichloromethane and concentrated underreduced pressure then extracted with ethyl acetate and water. Theorganic extracts were washed with brine, dried over magnesium sulfate,filtered, concentrated and purified by combiflash (EtOAc/Hex) to give3,5-bis(((tert-butyldimethylsilyl)oxy)methyl)-N-(2-(2-(2-methoxyethoxy)ethoxy)ethyl)-N-(2-methyl-2-(methyldisulfanyl)propyl)aniline(3.5 g, 40%). ¹H NMR (400 Hz, CDCl₃): δ 6.73 (s, 2H), 6.59 (s, 1H), 4.56(s, 4H), 3.65-3.51 (m, 14H), 3.30 (s, 3H), 2.38 (s, 3H), 1.28 (s, 6H),0.84 (s, 18H), 0.00 (s, 12H). MS (m/z): found 684.2 (M+Na)⁺. See FIG.46.

(5-((2-(2-(2-methoxyethoxy)ethoxy)ethyl)(2-methyl-2-(methyldisulfanyl)propyl)amino)-1,3-phenylene)dimethanol(1b)

Tetrabutylammonium fluoride (1M in THF) (10.57 ml, 10.57 mmol) was addeddropwise to stirring solution of3,5-bis(((tert-butyldimethylsilyl)oxy)methyl)-N-(2-(2-(2-methoxyethoxy)ethoxy)ethyl)-N-(2-methyl-2-(methyldisulfanyl)propyl)aniline(3.5 g, 5.29 mmol) in anhydrous THF (65 ml) at 0° C. in an ice bath.Following addition the mixture was stirred at ambient temperature fortwo hours. The mixture was quenched with saturated ammonium chloride andextracted with ethyl acetate. The extracts were washed with water andbrine, dried with magnesium sulfate, filtered and concentrated underreduced pressure. Purification by silica gel chromatography (MeOH/DCM)yielded(5-((2-(2-(2-methoxyethoxy)ethoxy)ethyl)(2-methyl-2-(methyldisulfanyl)propyl)amino)-1,3-phenylene)dimethanol(2 g, 87%). ¹H NMR (400 Hz, CDCl₃): δ 6.76 (s, 2H), 6.63 (s, 1H), 4.55(s, 4H), 3.65-3.51 (m, 14H), 3.35 (s, 3H), 2.44 (s, 3H), 1.33 (s, 6H);¹³C NMR (400 Hz, CDCl₃): δ 149.0, 142.35, 114.0, 111.1, 71.98, 70.7,70.6, 70.5, 67.6, 65.5, 59.75, 59.1, 53.9, 51.9, 26.6, 25.7, 20.75; MS(m/z): found 456.2 (M+Na)⁺. See FIG. 46.

Example 28

(5-(2-methyl-2-(methyldisulfanyl)propylamino)-1,3-phenylene)dimethanol

(5-amino-1,3-phenylene)dimethanol (2.5 g, 16.32 mmol) and2-(methyldithio)isobutyraldehyde (2.347 ml, 16.32 mmol) were stirred atambient temperature in absolute ethanol (82 ml) until completelydissolved (3 hours). The mixture was cooled to 0° C. in an ice bath andsodium borohydride (0.741 g, 19.59 mmol) was added. The reaction wasstirred for 1 hour at 0° C., and was then quenched slowly with cold 5%HCl solution. The mixture was diluted with dichloromethane and the pHwas adjusted to pH=8 with saturated sodium bicarbonate solution thenextracted with dichloromethane and then washed with brine. The organicextracts were dried over anhydrous sodium sulfate, filtered, andconcentrated under reduced pressure. Purification by silica gelchromatography (MeOH/DCM) yielded(5-(2-methyl-2-(methyldisulfanyl)propylamino)-1,3-phenylene)dimethanol(3 g, 65%) as a white solid. ¹H NMR (400 Hz, CDCl₃): δ6.62 (s, 1H), 6.54(s, 2H), 4.53 (s, 4H), 3.13 (s, 2H), 2.30 (s, 3H), 1.32 (s, 6H). SeeFIG. 47.

Example 29

tert-butyl9-hydroxy-8-methoxy-6-oxo-12a,13-dihydro-6H-benzo[5,6][1,4]diazepino[1,2-a]indole-11(12H)-carboxylate

To a solution of9-hydroxy-8-methoxy-11,12,12a,13-tetrahydro-6H-benzo[5,6][1,4]diazepino[1,2-a]indol-6-one3b (0.3 g, 1.012 mmol) in methanol (5.06 ml) were added di-tert-butyldicarbonate (0.265 g, 1.215 mmol), triethylamine (0.212 ml, 1.519 mmol)and DMAP (6.18 mg, 0.051 mmol). After 5 hours of stirring at ambienttemperature the reaction mixture was concentrated in vacuo. The residuewas redissolved in dichloromethane and filtered through celite.Purification by silica gel chromatography (20% EtOAc/DCM) yieldedtert-butyl9-hydroxy-8-methoxy-6-oxo-12a,13-dihydro-6H-benzo[5,6][1,4]diazepino[1,2-a]indole-11(12H)-carboxylate(0.21 g, 52%) as a white solid. ¹H NMR (400 Hz, CDCl₃): δ 8.25 (d, J=8.0Hz, 1H), 7.44 (s, 1H), 7.18 (t, J=7.2 Hz, 1H), 7.11 (d, J=7.2 Hz, 1H),6.98 (t, J=7.2 Hz, 1H), 6.39 (s, 1H), 4.37 (m, 1H), 3.75 (s, 3H), 3.42(m, 3H), 2.74 (dd, J=3.6, 16.4 Hz, 1H), 1.47 (s, 9H). See FIG. 48.

Example 30 Preparation and Testing of huMy9-6-31c

A reaction containing 2.0 mg/mL huMy9-6 antibody and 5 molar equivalentsof compound 31c (pretreated with 5-fold excess of sodium bisulfite in90:10 DMA:water) in 50 mM HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) pH 8.5 buffer and 10% v/v DMA(N,N-Dimethylacetamide) cosolvent was allowed to conjugate for 6 hoursat 25° C. Post-reaction, the conjugate was purified and buffer exchangedinto 250 mM Glycine, 10 mM Histidine, 1% sucrose, 0.01% Tween-20, 50 μMsodium bisulfite formulation buffer, pH 6.2, using NAP desalting columns(Illustra Sephadex G-25 DNA Grade, GE Healthcare). Dialysis wasperformed in the same buffer for 4 hours at room temperature utilizingSlide-a-Lyzer dialysis cassettes (ThermoScientific 20,000 MWCO).

The purified conjugate was found to have an average of 3.1 IGN moleculeslinked per antibody (by UV-Vis using molar extinction coefficientsε_(330 nm)=15,484 cm⁻¹M⁻¹ and ε_(280 nm)=30, 115 cm⁻¹M⁻¹ for 1, andε_(280 nm)=207,000 cm⁻¹M⁻¹ for My9-6 antibody), 98% monomer (by sizeexclusion chromatography), <0.2% unconjugated drug (by dual-columnreverse-phase HPLC analysis) and a final protein concentration of 0.4mg/ml.

In vitro potency measurements for conjugates of huMy9-6 with 31c at twodifferent drug loads were shown below. Both conjugates were highlypotent towards antigen-positive HL60-QC cells, with IC₅₀ values between1.3-1.8 pM. Antigen blocking with 1 μM unconjugated huMy9-6significantly diminished the potency, demonstrating the antigenspecificity of the cytotoxic effect.

IC₅₀ (pM) Conjugate huMy9-6 Specificity huMy9-6-31c IC₅₀ (pm) blockingwindow 3.1 IGN/Ab 1.8 940 522 3.9 IGN/Ab 1.3 790 608

Example 31 In Vivo Efficacy of Various Conjugates in Tumor Bearing NudeMice

In this study, the anti-tumor activity of several conjugates of theinvention are investigated in immune-compromised mice (nude or SCID),preferably female nude mice, bearing various tumors. In some cases, inaddition or as an alternative, nude rats may be employed. The conjugatesto be tested include any one or more of the conjugates described herein.The various tumor cell lines that can be used for inoculating the nudemice included HL60/QC, MOLM-13, NB4, HEL92.1.7, OCI-AML3, KB, and/or anyother cancer cell lines recognized in the art as a proper model for adisease indication (e.g., cancer). Some criteria that may be applied forthe selection of tumor cell lines suitable for in vivo evaluationinclude: a) expression of the target antigen on the tumor cell, and, b)sensitivity of tumor cells to the unconjugated drug in vitro. Forexample, an in vitro cell line sensitivity screen, such as the 60-cellline screen described by the U.S. National Cancer Institute (seeVoskoglou-Nomikos et al., 2003, Clinical Cancer Res. 9; 42227-4239,incorporated herein by reference) can be used as one of the guides todetermine the types of cancers that may be suitable for treatment withthe compounds of the invention. The potency of the various conjugatesagainst the various tumor cell lines, as expressed by IC₅₀ values (nM),is measured accordingly.

The various tumor cell lines are inoculated to nude or SCID mice usingsubstantially the same protocol as outlined in Example 15. For example,about 1×10⁶-5×10⁷ tumor cells (typically 1×10⁷) cells/mouse aresubcutaneously inoculated at a volume of approximately 0.1-0.2 mL/mouse,in the area over the right shoulder of female athymic nude mice, 6 weeksof age. When the tumor has reached an average size of ˜100 mm³(typically 6 to 8 days after tumor cell inoculation), mice arerandomized into groups (e.g., n=5-8 per group) by tumor volume.Treatment is initiated the day after randomization, and groups includesa control group dosed with the appropriate vehicle (200 μL/injection),or a single treatment at various doses (5 to 700 μg/kg) of the abovereferenced drug conjugates (50 μg/kg linked drug dose corresponded toabout 2 mg/kg antibody dose). Multiple dosing schedules (for exampletreatment at day 1, 3, 5, or day 1, 4, 7) may also be employed.

Median and mean tumor volume vs time is measured, with the datademonstrating a dose-dependent anti-tumor activity of the subjectconjugates. The minimum effective dose is then calculated and comparedto the maximum tolerated dose.

Example 32 Preparation of huMy9-6-Sulfo-SPDB-1d Using the4-nitroPy-Sulfo-SPDB Linker

A reaction containing 6 mg/mL huMy9-6 antibody and 5 molar equivalentsof the highly reactiveN-succinimidyl-4-(4-nitropyridyl-2-dithio)butanoate linker (20 mM stockin ethanol) was incubated for 3 h at 25° C. in 50 mM EPPS buffer at pH8. Unreacted linker was removed using a NAP desalting column (IllustraSephadex G-25 DNA Grade, GE Healthcare). The linker to antibody ratio(LAR) was determined to be about 2.3 based on antibody concentration andDTT-released nitropyridine-2-thione concentration by UV-Vis(ε_(394 nm)=14205 cm⁻¹M⁻¹ for 2-thio-4-nitropyridone).

Linker modified huMy9-6 was diluted to 2 mg/mL in 50 mM HEPES buffer atpH 8.5, 10% v/v DMA, and reacted with 2 molar equivalents of compound 1dper linker (5 mM stock in DMA; 4.6 equivalents per antibody) for 30 minat 25° C. Completion of disulfide exchange reaction was determined bymonitoring absorbance increase at 394 nm by UV.

Post-reaction, the conjugate was purified and buffer exchanged into 250mM glycine, 10 mM histidine, 1% sucrose, 0.01% Tween-20, 50 μM sodiumbisulfite at pH 6.2 using a desalting column (G-25 Sephadex, fine grade,GE Healthcare).

The purified conjugate was found to have an average of 2.1 molecules of1d linked per antibody (by UV-Vis using molar extinction coefficientsε_(330 nm)=15,484 cm⁻¹M⁻¹ and £_(280 nm)=30, 115 cm⁻¹M⁻¹ for 1d, andε_(280 nm)=207,000 cm⁻¹M⁻¹ for huMy9-6), 98% monomer (by size exclusionchromatography), <1% unconjugated 1d (by acetoneextraction/reverse-phase HPLC), a 70% protein yield, and a 32% overall1d yield. See FIG. 60.

1-51. (canceled)
 52. A conjugate comprising: a cytotoxic compound and acell binding agent (CBA), wherein the cytotoxic compound comprises alinking group which covalently links the cytotoxic compound to the CBA,and wherein said cytotoxic compound is represented by any one of thefollowing formulas:

or a pharmaceutically acceptable salt thereof, wherein: the double line

between N and C represents a single bond or a double bond, provided thatwhen it is a double bond X is absent and Y is —H, or a linear orbranched alkyl having 1 to 4 carbon atoms, and when it is a single bond,X is —H, the linking group, or an amine protecting moiety; Y is —H or aleaving group selected from —OR, —OCOR′, —OCOOR′, —OCONR′R″, —NR′R″,—NR′COR″, —NR′NR′R″, an optionally substituted 5- or 6-memberednitrogen-containing heterocycle, a guanidinum represented by—NR′(C═NH)NR′R″, an amino acid, or a peptide represented by —NRCOP′,wherein P′ is an amino acid or a polypeptide containing between 2 to 20amino acid units, —SR, —SOR′, —SO₂M, —SO₃M, —OSO₃M, halogen, cyano andan azido; or, Y is a sulfite (HSO₃, HSO₂ or a salt of HSO₃ ⁻, SO₃ ²⁻orHSO₂ ⁻ formed with a cation), metabisulfite (H₂S₂O₅ or a salt of S₂O₅ ²⁻formed with a cation), mono-, di-, tri-, and tetra-thiophosphate(PO₃SH₃, PO₂S₂H₂, POS₃H₂, PS₄H₂ or a salt of PO₃S³, PO₂S₂ ³ POS₃ ³⁻ orPS₄ ³⁻ formed with a cation), thio phosphate ester (R^(i)O)₂PS(OR^(i)),R^(i)SO, R^(i)SO₂, R^(i)SO₃, thiosulfate (HS₂O₃ or a salt of S₂O₃²⁻formed with a cation), dithionite (HS₂O₄ or a salt of S₂O₄ ²⁻ formedwith a cation), phosphorodithioate P(═S)(OR^(k′))(S)(OH) or a saltthereof formed with a cation), hydroxamic acid (R^(k′)(═O)NOH or a saltformed with a cation), formaldehyde sulfoxylate (HOCH₂SO₂ ⁻ or a salt ofHOCH₂SO₂ ⁻ formed with a cation, such as HOCH₂SO₂ ⁻Na⁺) or a mixturethereof, wherein R^(i) is a linear or branched alkyl having 1 to 10carbon atoms and is substituted with at least one substituent selectedfrom —N(R^(j))₂, —CO₂H, —SO₃H, and —PO₃H; R^(i) can be furtheroptionally substituted with a substituent for an alkyl described herein;R^(j) is a linear or branched alkyl having 1 to 6 carbon atoms; R^(k′)is a linear, branched or cyclic alkyl, alkenyl or alkynyl having 1 to 10carbon atoms, aryl, heterocyclyl or heteroaryl; M is —H or a cation; R,for each occurrence, is independently selected from the group consistingof —H, an optionally substituted linear, branched or cyclic alkyl,alkenyl or alkynyl having from 1 to 10 carbon atoms, a polyethyleneglycol unit —(CH₂CH₂O)_(n)—R^(c), an optionally substituted aryl having6 to 18 carbon atoms, an optionally substituted 5- to 18-memberedheteroaryl ring containing one or more heteroatoms independentlyselected from nitrogen, oxygen, and sulfur, or an optionally substituted3- to 18-membered heterocyclic ring containing 1 to 6 heteroatomsindependently selected from O, S, N and P; R′ and R″ are eachindependently selected from —H, —OH, —OR, —NHR, —NR₂, —COR, anoptionally substituted linear, branched or cyclic alkyl, alkenyl oralkynyl having from 1 to 10 carbon atoms, a polyethylene glycol unit—(CH₂CH₂O)_(n)—R^(c), and an optionally substituted 3-18-memberedheterocyclic ring having 1 to 6 heteroatoms independently selected fromO, S, N and P; R^(c) is —H or a substituted or unsubstituted linear orbranched alkyl having 1 to 4 carbon atoms, or the linking group; n is aninteger from 1 to 24; W is selected from C═O, C═S, CH₂, BH, SO and SO₂;X′ is selected from —H, an amine-protecting group, the linking group, anoptionally substituted linear, branched or cyclic alkyl, alkenyl oralkynyl having from 1 to 10 carbon atoms, a polyethylene glycol unit—(CH₂CH₂O)_(n)—R^(c), an optionally substituted aryl having 6 to 18carbon atoms, an optionally substituted 5- to 18-membered heteroarylring containing one or more heteroatoms independently selected fromnitrogen, oxygen, and sulfur, and an optionally substituted 3- to18-membered heterocyclic ring containing 1 to 6 heteroatomsindependently selected from O, S, N and P; Y′ is selected from —H, anoxo group, the linking group, an optionally substituted linear, branchedor cyclic alkyl, alkenyl or alkynyl having from 1 to 10 carbon atoms, anoptionally substituted 6- to 18-membered aryl, an optionally substituted5- to 18-membered heteroaryl ring containing one or more heteroatomsindependently selected from nitrogen, oxygen, and sulfur, an optionallysubstituted 3 to 18-membered heterocyclic ring having 1 to 6heteroatoms; R₁, R₂, R₃, R₄, R₁′, R₂′, R₃′ and R₄′ are eachindependently selected from the group consisting of —H, an optionallysubstituted linear, branched or cyclic alkyl, alkenyl or alkynyl havingfrom 1 to 10 carbon atoms, a polyethylene glycol unit—(OCH₂CH₂)_(n)—R^(c), halogen, guanidinium [—NH(C═NH)NH₂], —OR, —NR′R″,—NO₂, —NCO, —NR′COR″, —SR, a sulfoxide represented by —SOR′, a sulfonerepresented by —SO₂R′, a sulfonate —SO₃ ⁻M⁺, a sulfate —OSO₃ ⁻M⁺, asulfonamide represented by —SO₂NR′R″, cyano, an azido, —COR′, —OCOR′,—OCONR′R″ and the linking group; R₆ is —H, —R, —OR, —SR, —NR′R″, —NO₂,halogen or the linking group; Z and Z′ are independently selected from—(CH₂)_(n′)—, —(CH₂)_(n′)—CR₇R₈—(CH₂)_(na′)—,—(CH₂)_(n′)—NR₉—(CH₂)_(na′)—, —(CH₂)_(n′)—O—(CH₂)_(na′)— and—(CH₂)_(n′)—S—(CH₂)_(na′)—; n′ and na′ are the same or different, andare selected from 0, 1, 2 and 3; R₇ and R₈ are the same or different,and are each independently selected from —H, OH, —SH, —COOH, —NHR′, apolyethylene glycol unit —(OCH₂CH₂)_(n)—, an amino acid, a peptide unitbearing 2 to 6 amino acids, an optionally substituted linear, branchedor cyclic alkyl having from 1 to 10 carbon atoms; R₉ is independentlyselected from —H, an optionally substituted linear, branched or cyclicalkyl having from 1 to 10 carbon atoms, a polyethylene glycol unit—(OCH₂CH₂)_(n)—; A and A′ are the same or different, and areindependently selected from —O—, oxo (—C(═O)—), —CRR′O—, —CRR′—, —S—,—CRR′S—, —N(R₅)— and —CRR′N(R₅)—, R₅ for each occurrence isindependently —H or an optionally substituted linear or branched alkylhaving 1 to 10 carbon atoms; D and D′ are the same or different, and areindependently absent or selected from the group consisting of anoptionally substituted linear, branched or cyclic alkyl, alkenyl oralkynyl having 1 to 10 carbon atoms, an amino acid, a peptide bearing 2to 6 amino acids, and a polyethylene glycol unit (—OCH₂CH₂)_(n)—; L isabsent, the linking group, a polyethylene glycol unit (—OCH₂CH₂)_(n)—, alinear, branched or cyclic alkyl or alkenyl having 1 to 10 carbon atoms,a phenyl group, a 3- to 18-membered heterocyclic ring or a 5- to18-membered heteroaryl ring having 1 to 6 heteroatoms independentlyselected from O, S, N and P, wherein the alkyl or alkenyl is optionallysubstituted with the linking group; phenyl or heterocyclic or heteroarylring can be optionally substituted, wherein the substituent can be thelinking group; wherein the compound is not any one of the followingcompounds:

53-55. (canceled)
 56. The conjugate of claim 52, wherein the compound isrepresented by any one of the following formulas:

wherein: L′, L″, and L′″ are the same or different, and areindependently selected from —H, an optionally substituted linear,branched or cyclic alkyl, alkenyl or alkynyl having from 1 to 10 carbonatoms, a polyethylene glycol unit —(OCH₂CH₂)_(n)—R^(c), halogen,guanidinium [—NH(C═NH)NH₂], —OR, —NR′R″, —NO₂, —NR′COR″, —SR, asulfoxide represented by —SOR′, a sulfone represented by —SO₂R′, asulfonate —SO₃M, a sulfate —OSO₃M, a sulfonamide represented by—SO₂NR′R″, cyano, an azido, —COR′, —OCOR′, —OCONR′R″ and the linkinggroup, provided only one of L′, L″, and L′″ is the linking group; and Gis selected from —CH— or —N—.
 57. (canceled)
 58. The conjugate of claim56, wherein L′ is the linking group, and L″ and L′″ are —H. 59.(canceled)
 60. The conjugate of claim 58, wherein L′ is represented bythe following formula:—W′—R^(x)—V—R^(y)-J, wherein: W′ and V are the same or different, andare each independently absent, or selected from —CR^(e)R^(e)—, —O—,—O—C(═O)—, —C(═O)—O—, —S—, —SO—, —SO₂—, —CH₂—S—, —CH₂O—, —CH₂NR^(e)—,—O—(C═O)O—, —O—(C═O)N(R^(e))—, —N(R^(e))—, —N(R^(e))—C(═O)—,—C(═O)—N(R^(e))—, —N(R^(e))—C(═O)O—, —N(C(═O)R^(e))C(═O)—,—N(C(═O)R^(e))—, —(O—CH₂—CH₂)_(n)—, —SS—, or —C(═O)—, or an amino acid,or a peptide having 2 to 8 amino acids; R^(x) and R^(y) are the same ordifferent, and are each independently absent or an optionallysubstituted linear, branched or cyclic alkyl, alkenyl, or alkynyl having1 to 10 carbon atoms, an aryl bearing 6 to 10 carbon atoms or a 3- to8-membered hetereocyclic ring bearing 1 to 3 heteroatoms selected fromO, N or S; R^(e) and R^(e′) are the same or different, and are selectedfrom —H, a linear, branched or cyclic alkyl, alkenyl, or alkynyl having1 to 10 carbon atoms or —(CH₂—CH₂—O)_(n)—R^(k), wherein R^(k) is a —H, alinear, branched cyclic alkyl having 1 to 6 carbon atoms, optionallybearing a secondary amino (, —NHR¹⁰¹) or tertiary amino (—NR¹⁰¹R¹⁰²)group or a 5- or 6-membered nitrogen containing heterocycle, such aspiperidine or morpholine, wherein R¹⁰¹ and R¹⁰² are each independently alinear, branched, or cyclic alkyl, alkenyl or alkynyl having 1 to 10carbon atoms; preferably, R¹⁰¹ and R¹⁰² are each independently a linearor branched alkyl having 1 to 6 carbon atoms; n is an integer from 1 to24; and J is covalently linked to the CBA, and is selected from asuccinimide, a acetamido, —S—, —SS—, —CH₂S—, —CH(Me)S—, —C(Me)₂S—,—NR^(c1)—, —CH₂NR^(c1)—, —NR^(c1)N—, and —C(═O)—, wherein R^(c1) is —Hor a substituted or unsubstituted linear or branched alkyl having 1 to 4carbon atoms.
 61. The conjugate of claim 60, wherein J is —S—, —SS—, asuccinimide, or —C(═O)—. 62-65. (canceled)
 66. The conjugate of claim60, wherein: W′ is —O—, —N(R^(e))— or —N(R^(e))—C(═O)—; R^(e) is —H,-Me, or —(CH₂—CH₂—O)_(n)-Me; n is an integer from 2 to 6; R^(x) islinear or branched alkyl bearing 1 to 6 carbon atoms; V and R^(y) areabsent; and J is —C(═O)—.
 67. The conjugate of claim 60, wherein L′ isrepresented by the following formula:—W′—[CR_(1″)R_(2″)]_(a)—V—[Cy]₀₋₁—[CR_(3″)R_(4″)]_(b)—C(═O)—, wherein:R_(1″), R_(2″), and R_(3″) are each independently —H or -Me; R_(4″) is—H, -Me, —SO₃H, or —SO₃ ⁻M⁺, wherein M⁺ is a pharmaceutically acceptablecation; a is an integers from 0-2, b is an integer from 0-3; and, Cy isan optionally substituted 5-membered heterocyclic ring bearing an Nheteroatom, preferably Cy is


68. The conjugate of claim 60, wherein W′ is —N(R^(e))—; R^(e) is—(CH₂—CH₂—O)₂₋₆—R^(k), wherein R^(k) is a linear or branched alkylhaving 1 to 6 carbon atoms; and V is —S— or —SS—. 69-70. (canceled) 71.The conjugate of claim 60, wherein L′ is represented by the followingformula:NR^(e)—[CR_(1″)R_(2″)]_(a)—S—[CR_(3″)R_(4″)]_(b)—C(═O)—.
 72. Theconjugate of claim 60, which is:

wherein r is an integer from 1 to 10, Y is —H or —SO₃M, and M is —H or apharmaceutically acceptable cation.
 73. The conjugate of claim 60,wherein the antibody is huMy9-6.
 74. The conjugate of claim 60, whereinL′ is represented by the following formula:NR^(e)—[CR_(1″)R_(2″)]_(a)—S—Cy-[CR_(3″)R_(4″)]_(b)—C(═O)—.
 75. Theconjugate of claim 60, which is:

wherein r is an integer from 1 to 10, Y is —H or —SO₃M, and M is —H or apharmaceutically acceptable cation.
 76. The conjugate of claim 60,wherein the antibody is huMy9-6.
 77. The conjugate of claim 56, whereinthe compound is represented by the following formula:

wherein: W′ is absent, or selected from —O—, —N(R^(e))—,—N(R^(e))—C(═O)—, —N(C(═O)R^(e))—, —S—, —CH₂—S—, or —CH₂NR^(e)—; R^(x)is absent or selected from a linear, branched or cyclic alkyl having 1to 10 carbon atoms; R^(e) is —H, a linear, branched or cyclic alkyl,alkenyl or alkynyl having 1 to 10 carbon atoms or—(CH₂—CH₂—O)_(n)—R^(k), wherein R^(k) is a —H, a linear, branched cyclicalkyl having 1 to 6 carbon atoms, optionally bearing a secondary aminoor tertiary amino (NR¹⁰¹R¹⁰²) group or a 5 or 6-membered nitrogencontaining heterocycle, such as piperidine or morpholine, wherein R¹⁰¹and R¹⁰² are each independently a linear, branched, or cyclic alkyl,alkenyl or alkynyl having 1 to 10 carbon atoms; n is an integer from 2to 6; Z^(s) is linked to the CBA, and is selected from: a bond;

wherein: q is an integer from 1 to 5; and, M is —H or a cation, such asNa⁺ or K⁺.
 78. The conjugate of claim 77, wherein Z^(s) is:


79. The conjugate of claim 77, wherein W′ is —N(R^(e))—R^(e) is—(CH₂—CH₂—O)_(n)—R^(k), wherein R^(k) is a —H, a linear, branched, orcyclic alkyl having 1 to 6 carbon atoms.
 80. (canceled)
 81. Theconjugate of claim 79, wherein R^(k) is —H or -Me, n is 4, and q is 2.82. The conjugate of claim 81, wherein R^(x) is a linear or branchedalkyl having 1 to 6 carbon atoms.
 83. The conjugate of claim 81, whereinR^(x) is —(CH₂)_(p)—(CR^(f)R^(g))—, wherein R^(f) and R^(g) are eachindependently selected from H or a linear or branched alkyl having 1 to4 carbon atoms; and p is 0, 1, 2 or
 3. 84. The conjugate of claim 83,wherein R^(f) and R^(g) are the same or different, and are selected fromH and Me; and p is
 1. 85. The conjugate of claim 77, wherein: the doubleline

between N and C represents a single bond or a double bond, provided thatwhen it is a double bond X is absent and Y is —H, and when it is asingle bond, X is —H; Y is —OH or —SO₃M; M is —H or a pharmaceuticallyacceptable cation; X′ and Y′ are both —H; A and A′ are both —O—; R₆ is—OMe; and R^(x) is a linear or branched alkyl having 1 to 6 carbonatoms.
 86. The conjugate of claim 52, wherein the double line

between N and C represents a double bond.
 87. The conjugate of claim 52,wherein the double line

between N and C represents a single bond, X is —H, the linking group, oran amine protecting group; and Y is selected from —H, —OR, —OCOR′, —SR,—NR′R,″ an optionally substituted 5 or 6-membered nitrogen-containingheterocycle, —SO₃M, —SO₂M and a sulfate —OSO₃M.
 88. The conjugate ofclaim 87, wherein Y is selected from —H, —SO₃M, —OH, —OMe, —OEt or—NHOH.
 89. (canceled)
 90. The conjugate of claim 87, wherein M is —H,Na⁺ or K⁺.
 91. The conjugate of claim 52, wherein W, when present, isC═O, and, Z and Z′, when present, are —CH₂—. 92-103. (canceled)
 104. Theconjugate of claim 52, wherein: the double line

between N and C represents a double bond; Y is —H; W is C═O; R₁, R₂,R₁′, R₂′, R₄ and R₄′ are —H; one of R₃, or R₃′ is optionally the linkinggroup and the other is —H; R₆ is —OMe; Z and Z′ are —CH₂—; X′ is —H; Y′is —H; and A and A′ are —O—.
 105. The conjugate of claim 52, wherein theconjugate comprises 1-10 cytotoxic compounds, each cytotoxic compoundcomprising the linking group linking the cytotoxic compound to the CBA,and each cytotoxic compound on the conjugate is the same.
 106. Theconjugate of claim 52, wherein the cell-binding agent binds to targetcells selected from tumor cells, virus infected cells, microorganisminfected cells, parasite infected cells, autoimmune cells, activatedcells, myeloid cells, activated T-cells, B cells, or melanocytes; cellsexpressing the CD4, CD6, CD19, CD20, CD22, CD30, CD33, CD37, CD38, CD40,CD44, CD56, EpCAM, CanAg, CALLA, or Her-2 antigens; Her-3 antigens; orcells expressing insulin growth factor receptor, epidermal growth factorreceptor, and folate receptor.
 107. The conjugate of claim 106, whereinthe cell-binding agent is an antibody, a single chain antibody, anantibody fragment that specifically binds to the target cell, amonoclonal antibody, a single chain monoclonal antibody, or a monoclonalantibody fragment that specifically binds to a target cell, a chimericantibody, a chimeric antibody fragment that specifically binds to thetarget cell, a domain antibody, a domain antibody fragment thatspecifically binds to the target cell, a lymphokine, a hormone, avitamin, a growth factor, a colony stimulating factor, or anutrient-transport molecule.
 108. The conjugate of claim 107, whereinthe antibody is a resurfaced antibody, a resurfaced single chainantibody, a resurfaced antibody fragment, a monoclonal antibody, asingle chain monoclonal antibody, a monoclonal antibody fragmentthereof, a humanized antibody, a humanized single chain antibody, or ahumanized antibody fragment. 109-110. (canceled)
 111. A pharmaceuticalcomposition comprising the conjugate of claim 52 and a pharmaceuticallyacceptable carrier.
 112. A method of inhibiting abnormal cell growth ortreating a proliferative disorder, an autoimmune disorder, destructivebone disorder, infectious disease, viral disease, fibrotic disease,neurodegenerative disorder, pancreatitis or kidney disease in a mammalcomprising administering to said mammal a therapeutically effectiveamount of a conjugate of claim 52, and, optionally, a chemotherapeuticagent.
 113. (canceled)
 114. A method of inhibiting abnormal cell growthor treating a proliferative disorder, an autoimmune disorder,destructive bone disorder, infectious disease, viral disease, fibroticdisease, neurodegenerative disorder, pancreatitis or kidney disease in amammal comprising administering to said mammal a therapeuticallyeffective amount of a compound and, optionally, a chemotherapeuticagent, wherein the compound is of the following formula:

wherein Y is —H or —SO₃M, and M is —H or a pharmaceutically acceptablecation. 115-118. (canceled)